Self-buffering protein formulations

ABSTRACT

The invention herein described, provides, among other things, self-buffering protein formulations. Particularly, the invention provides self-buffering pharmaceutical protein formulations that are suitable for veterinary and human medical use. The self-buffering protein formulations are substantially free of other buffering agents, stably maintain pH for the extended time periods involved in the distribution and storage of pharmaceutical proteins for veterinary and human medical use. The invention further provides methods for designing, making, and using the formulation. In addition to other advantages, the formulations avoid the disadvantages associated with the buffering agents conventionally used in current formulations of proteins for pharmaceutical use. The invention in these and other respects can be productively applied to a wide variety of proteins and is particularly useful for making and using self-buffering formulations of pharmaceutical proteins for veterinary and medical use, especially, in particular, for the treatment of diseases in human subjects.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims full prioritybenefit of U.S. Provisional Application Ser. No. 60/690,582 filed 14Jun. 2005, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the formulation of proteins, especiallypharmaceutical proteins. In particular, it relates to self-bufferingbiopharmaceutical protein compositions, and to methods for designing,making, and using the compositions. It further relates to pharmaceuticalprotein compositions for veterinary and/or for human medical use, and tomethods relating thereto.

BACKGROUND OF THE INVENTION

Many aspects of pharmaceutical production and formulation processes arepH sensitive. Maintaining the correct pH of a finished pharmaceuticalproduct is critical to its stability, effectiveness, and shelf life, andpH is an important consideration in designing formulations foradministration that will be acceptable, as well as safe and effective.

To maintain pH, pharmaceutical processes and formulations use one ormore buffering agents. A variety of buffering agents are available forpharmaceutical use. The buffer or buffers for a given application mustbe effective at the desired pH. They must also provide sufficient buffercapacity to maintain the desired pH for as long as necessary. A goodbuffer for a pharmaceutical composition must satisfy numerous otherrequirements as wall. It must be appropriately soluble. It must not formdeleterious complexes with metal ions, be toxic, or unduly penetrate,solubilize, or absorb on membranes or other surfaces. It should notinteract with other components of the composition in any manner whichdecreases their availability or effectiveness. It must be stable andeffective at maintaining pH over the range of conditions to which itwill be exposed during formulation and during storage of the product. Itmust not be deleteriously affected by oxidation or other reactionsoccurring in its environment, such as those that occur in the processingof the composition in which it is providing the buffering action. Ifcarried over or incorporated into a final product, a buffering agentmust be safe for administration, compatible with other components of thecomposition over the shelf-life of the product, and acceptable foradministration to the end user.

Although there are many buffers in general use, only a limited numberare suitable for biological applications and, of these, fewer still areacceptable for pharmaceutical processes and formulations. As a result,it often is challenging to find a buffer that not only will be effectiveat maintaining pH but also will meet all the other requirements for agiven pharmaceutical process, formulation, or product.

The challenge of finding a suitable buffer for pharmaceutical use can beespecially acute for pharmaceutical proteins. The conformation andactivity of proteins are critically dependent upon pH. Proteins aresusceptible to a variety of pH sensitive reactions that are deleteriousto their efficacy, typically many more than affect small molecule drugs.For instance, to mention just a few salient examples, the side chainamides of asparagine and glutamine are deaminated at low pH (less than4.0) and also at neutral or high pH (greater than 6.0). Aspartic acidresidues promote the hydrolysis of adjacent peptide bonds at low pH. Thestability and disposition of disulfide bonds is highly dependent on pH,particularly in the presence of thiols. Solubility, flocculation,aggregation, precipitation, and fibrillation of proteins are criticallydependent on pH. The crystal habit important to some pharmaceuticalformulations also is critically dependent on pH. And pH is also animportant factor in surface adsorption of many pharmaceutical peptidesand proteins.

Buffering agents that catalyze reactions that inactivate and/or degradeone or more other ingredients, moreover, cannot be used inpharmaceutical formulations. Buffers for pharmaceutical use must havenot only the buffer capacity required to maintain correct pH, but alsothey must not buffer so strongly that their administration deleteriouslyperturbs a subject's physiological pH. Buffers for pharmaceuticalformulations also must be compatible with typically complex formulationprocesses. For instance, buffers that sublime or evaporate, such asacetate and imidazole, generally cannot be relied upon to maintain pHduring lyophilization and in the reconstituted lyophilization product.Other buffers that crystallize out of the protein amorphous phase, suchas sodium phosphate, cannot be relied upon to maintain pH in processesthat require freezing.

Buffers used to maintain pH in pharmaceutical end-products also must benot only effective at maintaining pH but also safe and acceptable foradministration to the subject. For instance, several otherwise usefulbuffers, such as citrate at low or high concentration and acetate athigh concentration, are undesirably painful when administeredparenterally.

Some buffers have been found to be useful in the formulation ofpharmaceutical proteins, such as acetate, succinate, citrate, histidine(imidazole), phosphate, and Tris. They all have undesirable limitationsand disadvantages. And they all have the inherent disadvantage of beingan additional ingredient in the formulation, which complicates theformulation process, poses a risk of deleteriously affecting otheringredients, stability, shelf-life, and acceptability to the end user.

There is a need, therefore, for additional and improved methods ofmaintaining pH in the production and formulation of pharmaceuticals andin pharmaceutical compositions, particularly in the production andformulation of biopharmaceutical proteins and in biopharmaceuticalprotein compositions.

SUMMARY

Therefore, it is among the various objects and aspects of the inventionto provide, in certain of the preferred embodiments, proteinformulations comprising a protein, particularly pharmaceuticallyacceptable formulations comprising a pharmaceutical protein, that arebuffered by the protein itself, that do not require additional bufferingagents to maintain a desired pH, and in which the protein issubstantially the only buffering agent (i.e., other ingredients, if any,do not act substantially as buffering agents in the formulation).

In this regard and others, it is among the various objects and aspectsof the invention to provide, in certain preferred embodiments,self-buffering formulations of a protein, particularly of apharmaceutical protein, characterized in that the concentration of theformulated protein provides a desired buffer capacity.

It is further among the various objects and aspects of the invention toprovide, in certain of the particularly preferred embodiments,self-buffering protein formulations, particularly pharmaceutical proteinformulations, in which the total salt concentration is less than 150 mM.

It is further among the various objects and aspects of the invention toprovide, in certain of the particularly preferred embodiments,self-buffering protein formulations, particularly pharmaceutical proteinformulations, that further comprise one or more polyols and/or one ormore surfactants.

It is also further among the various objects and aspects of theinvention to provide, in certain of the particularly preferredembodiments, self-buffering formulations comprising a protein,particularly a pharmaceutical protein, in which the total saltconcentration is less than 150 mM, that further comprise one or moreexcipients, including but not limited to, pharmaceutically acceptablesalts; osmotic balancing agents (tonicity agents); surfactants, polyols,anti-oxidants; antibiotics; antimycotics; bulking agents;lyoprotectants; anti-foaming agents; chelating agents; preservatives;colorants; and analgesics.

It is additionally among the various objects and aspects of theinvention to provide, in certain preferred embodiments, self-bufferingprotein formulations, particularly pharmaceutical protein formulations,that comprise, in addition to the protein, one or more otherpharmaceutically active agents.

Various additional aspects and embodiments of the invention areillustratively described in the following numbered paragraphs. Theinvention is described by way of reference to each of the items setforth in the paragraphs, individually and/or taken together in anycombination. Applicant specifically reserves the right to assert claimsbased on any such combination.

1. A composition according to any of the following, wherein thecomposition has been approved for pharmaceutical use by a national orinternational authority empowered by law to grant such approvalpreferably the European Agency for the Evaluation of Medical Products,Japan's Ministry of Health, Labor and Welfare, China's State DrugAdministration, United States Food and Drug Administration, or theirsuccessor(s) in this authority, particularly preferably the UnitedStates Food and Drug Administration or its successor(s) in thisauthority.

2. A composition according to any of the foregoing or the following,wherein the composition is produced in accordance with goodmanufacturing practices applicable to the production of pharmaceuticalsfor use in humans.

3. A composition according to any of the foregoing or the following,comprising a protein, the protein having a buffer capacity per unitvolume per pH unit of at least that of approximately: 2.0 or 3.0 or 4.0or 5.0 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0or 75.0 or 100 or 125 or 150 or 260 or 250 or 300 or 350 or 400 or 500mM sodium acetate buffer in pure water over the range of pH 5.0 to 4.0or pH 5.0 to 5.5, preferably as determined in accordance with themethods described in Example 1 and 2, particularly preferably at least2.0 mM, especially particularly preferably at least 3.0 mM, veryespecially particularly preferably at least 4.0 mM or at least 5.0 mM,especially particularly preferably at least 7.5 mM, particularlypreferably at least 10 mM, preferably at least 20 mM.

4. A composition according to any of the foregoing or the followingwherein, exclusive of the buffer capacity of the protein, the buffercapacity per unit volume per pH unit of the composition is equal to orless than that of 1.0 or 1.5 or 2.0 or 3.0 or 4.0 or 5.0 mM sodiumacetate buffer in pure water over the range of pH 4.0 to 5.0 or pH 5.0to 5.5, preferably as determined in accordance with the methodsdescribed in Example 1 and 2, particularly preferably less than that of1.0 mM, very especially particularly preferably less than that of 2.0mM, especially particularly preferably less than that of 2.5 mM,particularly preferably less than that of 3.0 mM, preferably less thanthat of 5.0 mM.

5. A composition according to any of the foregoing or the followingcomprising a protein wherein over the range of plus or minus 1 pH unitfrom the pH of the composition, the buffer capacity of the protein is atleast approximately: 1.00 or 1.50 or 1.63 or 2.00 or 3.00 or 4.00 or5.00 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0 or75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or 500 or700 or 1,000 mEq per liter per pH unit, preferably at leastapproximately 1.00, particularly preferably 1.50, especiallyparticularly preferably 1.63, very especially particularly preferably2.00, very highly especially particularly preferably 3.00, veryespecially particularly preferably 5.0, especially particularlypreferably 10.0, particularly preferably 20.0.

6. A composition according to any of the foregoing or the followingcomprising a protein wherein over the range of plus or minus 1 pH unitfrom the pH of the composition, exclusive of the protein, the buffercapacity per unit volume per pH unit of the composition is equal to orless than that of 0.50 or 1.00 or 1.50 or 2.00 or 3.00 or 4.00 or 5.00or 6.50 or 8.00 or 10.0 or 20.0 or 25.0 mM sodium acetate buffer in purewater over the range pH 5.0 to 4.0 or pH 5.0 to 5.5, particularlypreferably determined in accordance with Example 1 and/or Example 2.

7. A composition according to any of the foregoing or the following,wherein over a range of plus or minus 1 pH unit from a desired pH, theprotein provides at least approximately 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the buffer capacity of thecomposition, preferably at least approximately 75%, particularlypreferably at least approximately 85%, especially particularlypreferably at least approximately 90%, very especially particularlypreferably at least approximately 95%, very highly especiallyparticularly preferably at least approximately 99% of the buffercapacity of the composition.

8. A composition according to any of the foregoing or the following,wherein the concentration of the protein is between approximately: 20and 400, or 20 and 300, or 20 and 250, or 20 and 200, or 20 and 150mg/ml, preferably between approximately 20 and 400 mg/ml, particularlypreferably between approximately 20 and 250, especially particularlybetween approximately 20 and 150 mg/ml.

9. A composition according to any of the foregoing or the following,wherein the pH maintained by the buffering action of the protein isbetween approximately: 3.5 and 8.0, or 4.0 and 6.0, or 4.0 and 5.5, or4.0 and 5.0, preferably between approximately 3.5 and 8.0, especiallyparticularly preferably approximately 4.0 and 5.5.

10. A composition according to any of the foregoing or the following,wherein the salt concentration is less than: 150 mM or 125 mM or 100 mMor 75 mM or 50 mM or 25 mM, preferably 150 mM, particularly preferably125 mM, especially preferably 100 mM, very particularly preferably 75mM, particularly preferably 50 mM, preferably 25 mM.

11. A composition according to any of the foregoing or the following,further comprising one or more pharmaceutically acceptable salts;polyols; surfactants, osmotic balancing agents; tonicity agents;anti-oxidants; antibiotics; antimycotics; bulking agents;lyoprotectants; anti-foaming agents; chelating agents; preservatives;colorants; analgesics; or additional pharmaceutical agents.

12. A composition according to any of the foregoing or the following,comprising one or more pharmaceutically acceptable polyols in an amountthat is hypotonic, isotonic, or hypertonic, preferably approximatelyisotonic, particularly preferably isotonic, especially preferably anyone or more of sorbitol, mannitol, sucrose, trehalose; or glycerol,particularly especially preferably approximately 5% sorbitol, 5%mannitol, 9% sucrose, 9% trehalose, or 2.5% glycerol, very especially inthis regard 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose, or 2.5%glycerol.

13. A composition according to any of the foregoing or the following,further comprising a surfactant, preferably one or more of polysorbate20, polysorbate 80, other fatty acid esters of sorbitan,polyethoxylates, and poloxamer 188, particularly preferably polysorbate20 or polysorbate 80, preferably approximately 0.001 to 0.1% polysorbate20 or polysorbate 80, very preferably approximately 0.002 to 0.02%polysorbate 20 or polysorbate 80, especially 0.002 to 0.02% polysorbate20 or polysorbate 80.

14. A composition according to any of the foregoing or the following,wherein the protein is a pharmaceutical agent and the composition is asterile formulation thereof suitable for treatment of a non-human or ahuman subject.

15. A composition according to any of the foregoing or the following,wherein the protein is a pharmaceutical agent effective to treat adisease and the composition is a sterile formulation thereof suitablefor administration to a subject for treatment thereof.

16. A composition according to any of the foregoing or the following,wherein the protein does not induce a significantly deleteriousantigenic response following administration to a subject.

17. A composition according to any of the foregoing or the following,wherein the protein does not induce a significantly deleterious immuneresponse following administration to a subject.

18. A composition according to any of the foregoing or the following,wherein the protein is a human protein.

19. A composition according to any of the foregoing or the following,wherein the protein is a humanized protein.

20. A method according to any of the foregoing or the following, whereinthe protein is an antibody, preferably an IgA, IgD, IgE, IgG, or IgMantibody, particularly preferably an IgG antibody, very particularlypreferably an IgG1, IgG2, IgG3, or IgG4 antibody, especially an IgG2antibody.

21. A composition according to any of the foregoing or the following,wherein the protein comprises a: Fab fragment, Fab₂ fragment, Fab₃fragment, Fc fragment, say fragment, bis-scFv(s) fragment, minibody,diabody, triabody, tetrabody, VhH domain, V-NAR domain, V_(H) domain,V_(L) domain, camel Ig, Ig NAR, or peptibody, or a variant, derivative,or modification of any of the foregoing.

22. A composition according to any of the foregoing or the following,wherein the protein comprises an Fc fragment or a part thereof or aderivative or variant of an Fc fragment or part thereof.

23. A composition according to any of the foregoing or the following,wherein the protein comprises a first binding moiety of a pair ofcognate binding moieties, wherein the first moiety binds the secondmoiety specifically.

24. A composition according to any of the foregoing or the following,wherein the protein comprises (a) an Fc fragment or a part thereof or aderivative or variant of an Fe fragment or part thereof, and (b) a firstbinding moiety of a pair of cognate binding moieties.

25. A composition according to any of claim 1, 5, 7, 9, 11, 13, or 14,wherein the protein is selected from the group consisting of proteinsthat bind specifically to one or more CD proteins, HER receptor familyproteins, cell adhesion molecules, growth factors, nerve growth factors,fibroblast growth factors, transforming growth factors (TGF),insulin-like growth factors, osteoinductive factors, insulins andinsulin-related proteins, coagulation and coagulation-related proteins,colony stimulating factors (CSFs), other blood and serum proteins bloodgroup antigens; receptors, receptor-associated proteins, growth hormonereceptors, T-cell receptors; neurotrophic factors, neurotrophins,relaxins, interferons, interleukins, viral antigens, lipoproteins,integrins, rheumatoid factors, immunotoxins, surface membrane proteins,transport proteins, homing receptors, addressins, regulatory proteins,and immunoadhesins,

26. A composition according to any of the foregoing or the following,wherein the protein is selected from the group consisting of: OPGLspecific binding proteins, myostatin specific binding proteins, IL-4receptor specific binding proteins, IL1-R1 specific binding proteins,Ang2 specific binding proteins, NGF-specific binding proteins, CD22specific binding proteins, IGF-1 receptor specific binding proteins,B7RP-1 specific binding proteins, IFN gamma specific binding proteins,TALL-1 specific binding proteins, stem cell factors, Flt-3 ligands, andIL-17 receptors.

27. A composition according to any of the foregoing or the following,wherein the protein is selected from the group consisting of proteinsthat bind specifically to one or more of: CD3, CD4, CD8, CD19, CD20,CD34; HER2, HER3, HER4, the EGF receptor; LFA-1, Mol, p150,95, VLA-4,ICAM-1, VCAM, alpha v/beta 3 integrin; vascular endothelial growthfactor (“VEGF”); growth hormone, thyroid stimulating hormone, folliclestimulating hormone, luteinizing hormone, growth hormone releasingfactor, parathyroid hormone, mullerian-inhibiting substance, humanmacrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO),NGF-beta, platelet-derived growth factor (PDGF), aFGF, bFGF, epidermalgrowth factor (EGF), TGF-alpha, TGF-beta1, TGF-beta2, TGF-beta3,TGF-beta4, TGF-beta5, IGF-I, IGF-II, des(1-3)-IGF-I (brain IGF-I),insulin, insulin A-chain, insulin B-chain, proinsulin, insulin-likegrowth factor binding proteins;, such as, among others, factor VIII,tissue factor, von Willebrands factor, protein C, alpha-1-antitrypsin,plasminogen activators, such as urokinase and tissue plasminogenactivator (“t-PA”), bombazine, thrombin, and thrombopoietin; M-CSF,GM-CSF, G-CSF, albumin, IgE, flk2/flt3 receptor, obesity (OB) receptor,bone-derived neurotrophic factor (BDNF), NT3, NT-4, NT-5, NT-6); relaxinA-chain, relaxin B-chain, prorelaxin; interferon-alpha, -beta, and-gamma; IL-1 to IL-10; AIDS envelope viral antigen; calcitonin,glucagon, atrial natriuretic factor, lung surfactant, tumor necrosisfactor-alpha and -beta, enkephalinase, RANTES, mousegonadotropin-associated peptide. Dnase, inhibin, and activin; protein Aor D, bone morphogenetic protein (BMP), superoxide dismutase, decayaccelerating factor (DAF).

28. A composition according to any of the foregoing or the following,wherein the protein is selected from the group consisting of: Actimmune(Interferon, gamma-1b), Activase (Alteplase), Aldurazme (Laronidase),Amevive (Alefacept), Avonex (Interferon beta-1a), BeneFIX (Nonacogalfa), Beromun (Tasonermin), Beatseron (Interferon-beta-1b), BEXXAR(Tositumomab), Tev-Tropin (Somatropin), Bioclate or RECOMBINATE(Recombinant), CEREZME (Imiglucerase), ENBREL (Etanercept), Eprex(epoetin alpha), EPOGEN/Procit (Epoetin alfa), FABRAZYME (Agalsidasebeta), Fasturtec/Elitek ELITEK (Rasburicase), FORTEO (Teriparatide),GENOTROPIN (Somatropin), GlucaGen (Glucagon), Glucagon (Glucagon, rDNAorigin), GONAL-F (follitropin alfa), KOGENATE FS (Octocog alfa),HERCEPTIN (Trastuzumab), HUMATROPE (SOMATROPIN), HUMIRA (Adalimumab),Insulin in Solution, INFERGEN® (Interferon alfacon-1), KINERET®(anakinra), Kogenate FS (Antihemophilic Factor), LEUKIN (SARGRAMOSTIMRecombinant human granulocyte-macrophage colony stimulating Factor(rhuGM-CSF)), CAMPATH (Alemtuzumab), RITUXAN® (Rituximab), TNKase(Tenecteplase), MYLOTARG (gemtuzumab ozogamicin), NATRECOR (nesiritide),ARANESP (darbepoetin alfa), NEULASTA (pegfilgrastim), NEUMEGA(oprelvekin), NEUPOGEN (Filgrastim), NORDITROPIN CARTRIDGES(Somatropin), NOVOSEVEN (Eptacog alfa), NUTROPIN AQ (somatropin),Oncaspar (pegaspargase), ONTAK (denileukin diftitox), ORTHOCLONE OKT(muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS(peginterferon alfa-2a), PROLEUKIN (Aldesleukin), PULMOZYME (dornasealfa), Retavase (Reteplase), REBETRON Combination Therapy containingREBETOL® (Ribavirin) and INTRON® A (Interferon alfa-2b), REBIF(interferon beta-1a), REFACTO (Antihemophilic Factor), REFLUDAN(lepirudin), REMICADE (infliximab), REOPRO (abciximab)ROFERON®-A(Interferon alfa-2a), SIMULECT (baasiliximab), SOMAVERT (Pegivisomant),SYNAGIS® (palivizumab), Stemben (Ancestim, Stem cell factor), THYROGEN,INTRON® A (Interferon alfa-2b), PEG-INTRON® (Peginterferon alfa-2b),XIGRIS® (Drotrecogin alfa activated), XOLAIR® (Omalizumab), ZENAPAX®(daelizumab), and ZEVALIN® (Ibritumomab Tiuxetan).

29. A composition according to any of the foregoing or the following,wherein the protein is Ab-hCD22 or a fragment thereof, or a variant,derivative, or modification of Ab-hCD22 or of a fragment thereof;Ab-hIL4R or a fragment thereof, or a variant, derivative, ormodification of Ab-hIL4R or of a fragment thereof; Ab-hOPGL, or afragment thereof, or a variant, derivative, or modification of Ab-hOPGLor of a fragment thereof, or Ab-hB7RP1 or a fragment thereof, or avariant, derivative, or modification of Ab-hB7RP1 or of a fragmentthereof.

30. A composition according to any of the foregoing or the following,wherein the protein is: Ab-hCD22 or Ab-hIL4R or Ab-hOPGL or Ab-hB7RP1.

31. A composition according to any of the foregoing or the followingcomprising a protein and a solvent, the protein having a buffer capacityper unit volume per pH unit of at least that of 4.0 mM sodium acetate inwater over the range of pH 4.0 to 5.0 or pH 5.0 to 5.5, particularly asdetermined by the methods described in Examples 1 and 2, wherein thebuffer capacity per unit volume of the composition exclusive of theprotein is equal to or less than that of 2.0 mM sodium acetate in waterover the same ranges preferably determined in the same way.

32. A composition according to any of the foregoing or the followingcomprising a protein and a solvent, wherein at the pH of the compositionthe buffer capacity of the protein is at least 1.63 mEq per liter for apH change of the composition of plus or minus 1 pH unit wherein thebuffer capacity of the composition exclusive of the protein is equal toor less than 0.81 mEq per liter at the pH of the composition for a pHchange of plus or minus 1 pH unit.

33. A lyophilate which upon reconstitution provides a composition inaccordance with any of the foregoing or the following.

14. A kit comprising in one or more containers a composition or alyophilate in accordance with any of the foregoing or the following, andinstructions regarding use thereof.

35. A process for preparing a composition or a lyophilate according toany of the foregoing or the following, comprising removing residualbuffer using a counter ion.

36. A process for preparing a composition or a lyophilate according toany of the foregoing or the following, comprising removing residualbuffer using any one or more of the following in the presence of acounter ion: chromatography, dialysis, and/or tangential flowfiltration.

37. A process for preparing a composition or a lyophilate according toany of the foregoing or the following, comprising removing residualbuffer using tangential flow filtration.

38. A process for preparing a composition or a lyophilate according toany of the foregoing or the following comprising a step of dialysisagainst a solution at a pH below that of the preparation, and, ifnecessary, adjusting the pH thereafter by addition of dilute acid ordilute base.

39. A method for treating a subject comprising administering to asubject in an amount and by a route effective for treatment acomposition according to any of the foregoing or the following,including a reconstituted lyophilate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts titration data and buffer capacity as a function ofconcentration for sodium acetate standard buffers over the range from pH5.0 to 4.0. Panel A is a graph that depicts the pH change upon acidtitration of several different concentrations of a standard sodiumacetate buffer, as described in Example 1. pH is indicated on thevertical axis. The amount of acid added to each solution is indicated onthe horizontal axis in microequivalents of HCl added per nil of solution(μEq/ml). The linear least squares trend lines are depicted for eachdataset. Acetate concentrations are indicated in the inset. Panel B is agraph that depicts the buffer capacity of the acetate buffers over theacidic pH range as determined from the titration data depicted in PanelA, as described in Example 1. Buffer capacity is indicated on thevertical axis as microequivalents of acid per ml of buffer solution perunit change in pH (μEq/ml-pH). Acetate concentration is indicated on thehorizontal axis in mM.

FIG. 2 depicts titration data and buffer capacity as a function ofconcentrations for sodium acetate standard buffers over the range frompH 5.0 to 5.5. Panel A is a graph that depicts the pH change upon basetitration of several different concentration of a standard sodiumacetate buffer, as described in Example 2. pH is indicated on thevertical axis. The amount of base added to each solution is indicated onthe horizontal axis in microequivalents of NaOH added per ml of solution(μEq/ml). The linear least squares trend lines are depicted for eachdataset. Acetate concentrations are indicated in the inset. Panel B is agraph that depicts the buffer capacity of the acetate buffers over thebasic pH range as determined from the titration data depicted in Panel Aand described in Example 2. Buffer capacity is indicated on the verticalaxis as microequivalents of base per ml of buffer solution per unitchange in pH (μEq/ml-pH). Acetate concentration is indicated on thehorizontal axis in mM.

FIG. 3 depicts the determination of acetate concentration in acetatebuffer standards, as described in Example 3. The graph shows a standardcurve for the determinations, with peak area indicated on the verticalaxis and the acetate concentration indicated on the horizontal axis. Thenominal and the measured amounts of acetate in the solutions used forthe empirical determination of buffer capacity are tabulated below thegraph.

FIG. 4 is a graph that depicts the pH change upon acid titration ofseveral different concentrations of Ab-hOPGL over the range of pH 5.0 to4.0, as described in Example 4. pH is indicated on the vertical axis.The amount of acid added to the solutions is indicated on the horizontalaxis in microequivalents of HCl added per ml of buffer solution(μEq/ml). The linear least squares trend lines are depicted for eachdataset. Ab-hOPGL concentrations are indicated in the inset.

FIG. 5 is a graph that depicts the pH change upon base titration ofseveral different concentrations of Ab-hOPGL over the range 5.0 to 6.0,as described in Example 5. pH is indicated on the vertical axis. Theamount of base added to the solutions is indicated on the horizontalaxis in microequivalents of NaOH added per ml of buffer solution(μEq/ml). The linear least squares trend lines are depicted for eachdataset. Ab-hOPGL concentrations are indicated in the inset.

FIG. 6 shows the residual acetate levels in Ab-hOPGL solutions used fordetermining buffer capacity. The graph shows the standard curve used forthe acetate determinations as described in Example 6. The nominal andthe experimentally measured acetate concentrations in the solutions aretabulated below the graph.

FIG. 7 is a graph depicting the buffer capacity of Ab-hOPGL plus orminus residual acetate in the pH range 5.0 to 4.0. The data wereobtained as described in Example 7. The upper line shows Ab-hOPGL buffercapacity with residual acetate. The lower line shows Ab-hOPGL buffercapacity adjusted for residual acetate. The vertical axis indicatesbuffer capacity in microequivalents of acid per ml of Ab-hOPGL solutionper unit of pH (μEq/ml-pH). The horizontal axis indicates theconcentration of Ab-hOPGL in mg/ml. The buffer capacities of differentconcentrations of standard acetate buffers as described in Example 1 areshown as horizontal lines. The concentrations of the buffers areindicated above the lines.

FIG. 8 is a graph depicting the buffer capacity of Ab-hOPGL plus orminus residual acetate in the basic pH range pH 5.0 to 6.0. The datawere obtained as described in Example 8. The upper line depicts Ab-hOPGLbuffer capacity with residual acetate. The lower line depicts Ab-hOPGLbuffer capacity adjusted for residual acetate. The vertical axisindicates buffer capacity in microequivalents of base added per ml ofbuffer solution per unit of pH (μEq/ml-pH). The horizontal axisindicates the concentration of Ab-hOPGL in mg/ml. The buffer capacitiesof several concentrations of standard sodium acetate buffers asdescribed in Example 2 are indicated by horizontal lines. The acetateconcentrations are indicated above each line.

FIG. 9 depicts, in a pair of charts, pH and Ab-hOPGL stability inself-buffering and conventionally buffered formulations. Panel A depictsthe stability of self-buffered Ab-hOPGL, Ab-hOPGL formulated in acetatebuffer, and Ab-hOPGL formulated in glutamate as a function of storagetime at 4° C. over a period of six months. The vertical axis indicatesAb-hOPGL stability in percent Ab-hOPGL monomer determined by SE-HPLC.Storage time is indicated on the horizontal axis. Panel B depicts the pHof the same three formulations measured over the same period of time.The determinations of protein stability and the measurements of pH aredescribed in Example 9.

FIG. 10 depicts titration curves and buffer capacities for severalconcentrations of self-buffering Ab-hB7RP1 formulations over the rangeof pH 5.0 to 4.0. Panel A shows the titration data. pH is indicated onthe vertical axis. The amount of acid added to the solutions isindicated on the horizontal axis in microequivalents of HCl added per mlof buffer solution (μEq/ml). The linear least squares trend lines aredepicted for each dataset. The Ab-hB7RP1 concentrations are indicated inthe inset. Panel B depicts the buffer capacities of Ab-hB7RP1formulations. The upper line shows the buffer capacities for theformulations including the contribution of residual acetate. The lowerline shows the buffer capacities for formulations after subtracting thecontribution of residual acetate based on SE-HPLC determinations asdescribed in Example 3. Linear least squares trend lines are shown forthe two data sets. The vertical axis indicates buffer capacity inmicroequivalents of acid per ml of buffer solution per unit of pH(μEq/ml-pH). The concentration of Ab-hB7RP1 is indicated on thehorizontal axis in mg/ml. The buffer capacities of severalconcentrations of standard sodium acetate buffers as described inExample 1 are shown by dashed horizontal lines. The acetate bufferconcentration are shown below each line. The results were obtained asdescribed in Example 10.

FIG. 11 depicts titration curves and buffer capacities for severalconcentrations of self-buffering Ab-hB7RP1, formulations over the rangeof pH 5.0 to 6.0. Panel A shows the titration data. pH is indicated onthe vertical axis. The amount of base added to the solutions isindicated on the horizontal axis in microequivalents of NaOH added perml of buffer solution (μEq/ml). The linear least squares trend lines aredepicted for each dataset. The Ab-hB7RP1 concentrations are indicated inthe inset. Panel B depicts the buffer capacities of Ab-hB7RP1formulations. The upper line shows the buffer capacities for theformulations containing residual acetate. The lower line shows thebuffer capacities for formulations adjusted to remove the contributionof residual acetate. Linear least squares trend lines are shown for thetwo data sets. The vertical axis indicates buffer capacity inmicroequivalents of base per ml of buffer solution per unit of pH(μEq/ml-pH). The concentration of Ab-hB7RP1 is indicated on thehorizontal axis in mg/ml. The buffer capacities of severalconcentrations of standard sodium acetate buffers as described inExample 2 are shown by dashed horizontal lines. The acetate bufferconcentrations are shown above each line. The results were obtained asdescribed in Example 11.

FIG. 12 depicts Ab-hB7RP1 stability in self-buffering and conventionallybuffered formulations at 4° C. and 29° C. Panel A depicts the stabilityof self-buffered Ab-hB7RP1, Ab-hB7RP1 formulated in acetate buffer, andAb-hB7RP1 formulated in glutamate as a function of storage at 4° C. overa period of six months. The vertical axis depicts Ab-hB7RP1 monomer inthe samples determined by SE-HPLC. Time is indicated on the horizontalaxis. Panel B depicts the stability of the same three formulations as afunction of storage at 29° C. over the same period of time. Axes inPanel B are the same as in Panel A. The determinations of proteinstability by HPLC-SE are described in Example 12.

FIG. 13 depicts pH stability in self buffer formulations of Ab-hB7RP1 at4° C. and 29° C. The vertical axis indicates pH. Time, in weeks, isindicated on the horizontal axis. Temperatures of the datasets areindicated in the inset. The data were obtained as described in Example13.

FIG. 14 depicts the buffer capacity of self-buffering formulations ofAb-hCD22 as a function of Ab-hCD22 concentration over the range of pH4.0 to 6.0. Panel A depicts the buffer capacities of self-bufferingAb-hCD22 formulations as a function of Ab-hCD22 concentration over therange of pH 4.0 to 5.0. Panel B depicts the buffer capacities ofself-buffering Ab-hCD22 formulations as a function of concentration overthe range of pH 5.0 to 6.0. In both panels the vertical axis indicatesbuffer capacity in microequivalents of base per ml of buffer solutionper unit of pH (μEq/ml-pH), and the horizontal axis indicates Ab-hCD22,concentrations in mg/ml. For reference, the buffer capacity of 10 mMsodium acetate as described in Example 1 is shown in both panels by adashed horizontal line. The results shown in the Figure were obtained asdescribed in Example 14.

FIG. 15 depicts titration curves and buffer capacities for severalconcentrations of self-buffering Ab-hIL4R formulations over the range ofpH 5.0 to 4.0. Panel A shows the titration data. pH is indicated on thevertical axis. The amount of acid added to the solutions is indicated onthe horizontal axis in microequivalents of HCl added per ml of buffersolution (μEq/ml). The linear least squares trend lines are depicted foreach dataset. The Ab-hIL4R concentrations are indicated in the inset.Panel B depicts the buffer capacities of Ab-hIL4R as a function ofconcentration. The linear least squares trend line is shown for thedataset. The vertical axis indicates buffer capacity in microequivalentsof base per ml of buffer solution per unit of pH (μEq/ml-pH). Theconcentration of Ab-hIL4R is indicated on the horizontal axis in mg/ml.The buffer capacities of several concentrations of standard sodiumacetate buffers as described in Example 1 are shown by dashed horizontallines. The acetate buffer concentrations are shown above each line. Theresults were obtained as described in Example 15.

FIG. 16 depicts titration curves and buffer capacities for severalconcentrations of self-buffering Ab-hIL4R formulations over the range ofpH 5.0 to 6.0. Panel A shows the titration data. pH is indicated on thevertical axis. The amount of base added to the solutions is indicated onthe horizontal axis in microequivalents of NaOH added per ml of buffersolution (μEq/ml). The linear least squares trend lines are depicted foreach dataset. The Ab-hIL4R concentrations are indicated in the inset.Panel B depicts the buffer capacities of Ab-hIL4R as a function ofconcentration. The linear least squares trend line is shown for thedataset. The vertical axis indicates buffer capacity in microequivalentsof base per ml of buffer solution per unit of pH (μEq/ml-pH). Theconcentration of Ab-hIL4R is indicated on the horizontal axis in mg/ml.The buffer capacities of several concentrations of standard sodiumacetate buffers as described in Example 2 are shown by dashed horizontallines. The acetate buffer concentrations are shown above each line. Theresults were obtained as described in Example 16.

FIG. 17 depicts Ab-hIL4R and pH stability in acetate buffered andself-buffered formulations of Ab-hIL4R at 37° C. as a function of time.Panel A is a bar graph showing Ab-hIL4R stability over four weeks at 37°C. The vertical axis indicates stability in per cent monomeric Ab-hIL4Ras determined by SE-HPLC. The horizontal axis indicates storage time inweeks. The insert identifies the data for the acetate and for theself-buffered formulations. Panel B shows the pH stability of the sameformulations for the same conditions and time periods. The pH isindicated on the vertical axis. Storage time in weeks is indicated onthe horizontal axis. Data for the acetate and self-buffered formulationsare indicated in the inset. The data were obtained as described inExample 17.

GLOSSARY

The meanings ascribed to various terms and phrases as used herein areillustratively explained below.

“A” or “an” herein means “at least one;” “one or more than one.”

“About,” unless otherwise stated explicitly herein, means ∀20%. Forinstance about 100 herein means 80 to 120, about 5 means 4 to 6, about0.3 means 0.24 to 0.36, and about 60% means 48% to 72% (not 40% to 80%).

“Agonist(s)” means herein a molecular entity that is different from acorresponding stimulatory ligand but has the same stimulatory effect.For instance (although agonists work through other mechanisms), for ahormone that stimulates an activity by binding to a correspondinghormone receptor, an agonist is a chemically different entity that bindsthe hormone receptor and stimulates its activity.

“Antagonist(s)” means herein a molecular entity that is different from acorresponding ligand and has an opposite effect. For instance (althoughantagonists work through other mechanisms), one type of antagonist of ahormone that stimulates an activity by binding to a correspondinghormone receptor is a chemical entity that is different from the hormoneand binds the hormone receptor but does not stimulate the activityengendered by hormone binding, and by this action inhibits the effectoractivity of the hormone.

“Antibody(s)” is used herein in accordance with its ordinary meaning inthe biochemical and biotechnological arts.

Among antibodies within the meaning of the term as it is used herein,are those isolated from biological sources, including monoclonal andpolyclonal antibodies, antibodies made by recombinant DNA techniques(also referred to at times herein as recombinant antibodies), includingthose made by processes that involve activating an endogenous gene andthose that involve expression of an exogenous expression construct,including antibodies made in cell culture and those made in transgenicplants and animals, and antibodies made by methods involving chemicalsynthesis, including peptide synthesis and semi-synthesis. Also withinthe scope of the term as it is used herein, except as otherwiseexplicitly set forth, are chimeric antibodies and hybrid antibodies,among others.

The prototypical antibody is a tetrameric glycoprotein comprised of twoidentical light chain-heavy chain dimers joined together by disulfidebonds. There are two types of vertebrate light chains, kappa and lambda.Each light chain is comprised of a constant region and a variableregion. The two light chains are distinguished by constant regionsequences. There are five types of vertebrate heavy chains: alpha,delta, epsilon, gamma, and mu. Each heavy chain is comprised of avariable region and three constant regions. The five heavy chain typesdefine five classes of vertebrate antibodies (isotypes): IgA, IgD, IgE,IgG, and IgM. Each isotype is made up of, respectively, (a) two alpha,delta, epsilon, gamma, or mu heavy chains, and (b) two kappa or twolambda light chains. The heavy chains in each class associate with bothtypes of light chains; but, the two light chains in a given molecule areboth kappa or both lambda. IgD, IgE, and IgG generally occur as “free”heterotetrameric glycoproteins. IgA and IgM generally occur in complexescomprising several IgA or several IgM heterotetramers associated with a“J” chain polypeptide. Some vertebrate isotypes are classified intosubclasses, distinguished from one another by differences in constantregion sequences. There are four human IgG subclasses, IgG1, IgG2, IgG3,and IgG4, and two IgA subclasses, IgA1 and IgA2, for example. All ofthese and others not specifically described above are included in themeaning of the term “antibody(s)” as used herein.

The term “antibody(s)” farther includes amino acid sequence variants ofany of the foregoing as described further elsewhere herein.

“Antibody-derived” as used herein means any protein produced from anantibody, and any protein of a design based on an antibody. The termincludes in its meaning proteins produced using all or part of anantibody, those comprising all or part of an antibody, and thosedesigned in whole or in part on the basis of all or part of an antibody.“Antibody-derived” proteins include, but are not limited to, Fc, Fab,and Fab₂ fragments and proteins comprising the same, V_(H) domain andV_(L) domain fragments and proteins comprising the same, other proteinsthat comprise a variable and/or a constant region of an antibody, inwhole or in part, scFv(s) intrabodies, maxibodies, minibodies,diabodies, amino acid sequence variants of the foregoing, and a varietyof other such molecules, including but not limited to others describedelsewhere herein.

“Antibody-related” as used herein means any protein or mimeticresembling in its structure, function, or design an antibody or any partof an antibody. Among “antibody-related” proteins as the term is usedherein are “antibody-derived” proteins as described above. It is to benoted that the terms “antibody-derived” and “antibody-related”substantially overlap; both terms apply to many such proteins. Examplesof “antibody-related” proteins, without implying limitation in thisrespect, are peptibodies and receptibodies. Other examples of“antibody-related” proteins are described elsewhere herein.

“Antibody polypeptide(s)” as used herein, except as otherwise noted,means a polypeptide that is part of an antibody, such as a light chainpolypeptide, a heavy chain polypeptide and a J chain polypeptide, tomention a few examples, including among others fragments, derivatives,and variants thereat and related polypeptides.

“Approximately” unless otherwise noted means the same as about.

“Binding moiety(s)” means a part of a molecule or a complex of moleculesthat binds specifically to part of another molecule or complex ofmolecules. The binding moiety may be the same or different from the partof the molecule or complex of molecules to which it binds. The bindingmoiety may be all of a molecule or complex of molecules as well.

“Binds specifically” is used herein in accordance with its ordinarymeaning in the art and means, except as otherwise noted, that binding isstronger with certain specific moieties than it is to other moieties ingeneral, that it is stronger than non-specific binding that may occurwith a wide variety of moieties, and that binding is selective forcertain moieties and does not occur to as strong an extent with others.In the extreme case of specific binding, very strong binding occurs witha single type of moiety, and there is no non-specific binding with anyother moiety.

“Co-administer” means an administration of two or more agents inconjunction with one another, including simultaneous and/or sequentialadministration.

“Cognate(s)” herein means complementary, fitting together, matching,such as, for instance, two jigsaw puzzles that fit one another, thecylinder mechanism of a lock and the key that opens it, the substratebinding site of an enzyme and the substrate of the enzyme, and a targetand target binding protein that binds specifically thereto.

“Cognate binding moieties” herein means binding moieties that bindspecifically to one another. Typically, but not always, it means a pairof binding moieties that bind specifically to one another. The moietiesresponsible for highly selective binding of a specific ligand and ligandreceptor provide an illustrative example of cognate binding moieties.Another example is provided by the moieties that binds an antigen and anantibody.

“Composition” means any composition of matter comprising one or moreconstituents, such as a formulation.

“Comprised of” is a synonym of “comprising” (see below).

“Comprising” means including, without further qualification, limitation,or exclusion as to what else may or may not be included. For example, “acomposition comprising x and y” means any composition that contains xand y, no matter what else it may contain. Likewise, “a methodcomprising x” is any method in which x is carried out, no matter whatelse may occur.

“Concentration” is used herein in accordance with its well-known meaningin the art to mean the amount of an item in a given amount of a mixturecontaining the item, typically expressed as a ratio. For example,concentration of a solute, such as a protein in a solution, can beexpressed in many ways, such as (but not limited to): (A) Weight Percent(i)=weight of solute per 100 units of solvent volume; (B) Weight Percent(ii)=weight of solute per 100 units of total weight; (C) Weight Percent(iii)=weight of solute per 100 units of solvent by weight; (D) MassPercent=mass of solute per 100 mass units of solution; (B) MoleFraction=moles of solute per total moles of all components; (F)Molarity=moles of solute per liter of solution (i.e., solute plussolvent); (G) Molality=moles of solute per Kg of solvent; and (H) VolumeMolality=moles of solute per liter of solvent.

“Control region(s)” is used herein in accordance with its well-knownmeaning in the art, and except as noted otherwise, refers to regions inDNA or proteins that are responsible for controlling one or morefunctions or activities thereof. For instance, “expression controlregion” with reference to the control of gene expression, means theregions in DNA that are required for transcription to occur properly andthat are involved in regulating when transcription occurs, howefficiently it occurs, when it is stopped, and the like.

“De novo” is used herein in accordance with its well-known meaning inthe art, to denote something made from new. For instance, a de novoamino acid sequence is one not derived from a naturally occurring aminoacid sequence, although, such a de novo sequence may have similaritieswith a naturally occurring sequence. De nova amino acid sequences can begenerated, for instance, by a priori design, by combinatorial methods,by selection methods. They can be made, for example, by chemicalsynthesis, by semi-synthesis, and by a variety of recombinant DNAtechniques, all of which are well know to those skilled in the art.

“Deleterious” means, as used herein, harmful. By way of illustration,“deleterious” processes include, for example, harmful effects of diseaseprocesses and harmful side effects of treatments.

“Derivative(s)” is used herein to mean derived from, in substance, form,or design, such as, for instance, a polypeptide that is based on butdiffers from a reference polypeptide, for instance, by alterations toits amino acid sequence, by fusion to another polypeptide, or bycovalent modification.

“Disease(s)” a pathology, a condition that deleteriously affects healthof a subject.

“Disorder(s)” a malediction, a condition that deleteriously altershealth.

“Dysfunction” means, as used herein, a disorder, disease, or deleteriouseffect of an otherwise normal process.

“Effective amount” generally means an amount which provides the desiredlocal or systemic effect. For example, an effective amount is an amountsufficient to effectuate a beneficial or desired clinical result. Theeffective amount can be provided all at once in a single administrationor in fractional amounts that provide the effective amount in severaladministrations. The precise determination of what would be consideredan effective amount may be based on factors individual to each subject,including their size, age, injury, and/or disease or injury beingtreated, and amount of time since the injury occurred or the diseasebegan. One skilled in the art will be able to determine the effectiveamount for a given subject based on these considerations which areroutine in the art. As used herein, “effective dose” means the same as“effective amount.”

“Effective route” generally means a route which provides for delivery ofan agent to a desired compartment, system, or location. For example, aneffective route is one through which an agent can be administered toprovide at the desired site of action an amount of the agent sufficientto effectuate a beneficial or desired clinical result.

“Endogenous” (such as endogenous gene) is used herein to refer to, forinstance, genes and other aspects of DNA, such as control regions, thatnaturally occur in a genome and organism, unless otherwise indicated.

“Exogenous” (such as exogenous gene), unless otherwise indicated, isused herein generally to mean, for instance, DNA from an outside source,such as DNA introduced to a cell and incorporated into its genome.

“FBS” means fetal bovine serum.

“Formulation(s)” means a combination of at least one active ingredientwith one or more other ingredients for one or more particular uses, suchas storage, further processing, sale, and/or administration to asubject, such as, for example, administration to a subject of a specificagent in a specific amount, by a specific route, to treat a specificdisease.

“Fragment(s)” herein means part of a larger entity, such as a part of aprotein; for instance, a polypeptide consisting of less than the entireamino acid sequence of a larger polypeptide. As used herein, the termincludes fragments formed by terminal deletion and fragments formed byinternal deletion, including those in which two or more non-contiguousportions of a polypeptide are joined together to form a smallerpolypeptide, which is a fragment of the original.

“Fusion protein(s)” herein, means a protein formed by fusing all or partof two polypeptides, which may be either the same or different. Typicalfusion proteins are made by recombinant DNA techniques, by end to endjoining of nucleotides encoding the two (or more) polypeptides.

“Genetically engineered” herein means produced using a deliberateprocess of genetic alteration, such as by recombinant DNA technology,classical methods of genetic manipulation, chemical methods, acombination of all three, or other methods.

“Homolog(s)” herein means having homology to another entity, such as aprotein that is homologous to another protein. Homologous meansresembling in structure or in function.

“Ionization” herein means the change of net charge on a substance by atleast one, including loss or gain of charge, such as the ionization ofacetic acid in low pH solution, from HOAc to OAc⁻ and H⁺.

“k” herein denotes an equilibrium co-efficient, in accordance with itsstandard meaning in chemistry.

“k_(a)” herein denotes the dissociation constant of a particularhydrogen of a molecule, in accordance with its standard meaning inchemistry, such as, for example, the dissociation constant of the acidichydrogen of acetic acid.

“k_(d)” herein denotes a dissociation constant of a pair of chemicalentities (or moieties), in accordance with its standard meaning inchemistry.

“Kit” means a collection of items used together for a given purpose orpurposes.

“Ligand(s)” herein means a molecular entity that binds selectively andstoichiometrically to one or more specific sites on one more othermolecular entities. Binding typically is non-covalent, but can becovalent as well. A very few examples, among many others, are (a)antigens, which typically bind non-covalently to the binding sites oncognate antibodies; (b) hormones, which typically bind hormonereceptors, non-covalently; (c) lectins, which bind specific sugars,non-covalently; (d) biotins, which bind multiple sites on avidin andother avidin-like proteins, non-covalently; (e) hormone antagonists,which bind hormone receptors and inhibit their activity and/or that ofthe corresponding hormone; and (f) hormone agonists, which similarlybind hormone receptors but stimulate their activity.

“Ligand-binding moiety(s)” herein means a molecular entity that binds aligand, typically, a part of a larger molecular entity that binds theligand, or a molecular entity derived therefrom.

“Ligand-binding protein(s)” herein means a protein that binds a ligand.

“Ligand moiety(s)” herein means a molecular entity that binds to aligand-binding molecular entity in much the same way as does thecorresponding ligand. A ligand moiety can be all of a ligand, or part ofit, derived from a ligand, or generated de novo. Typically, however, theligand moiety is more or less exclusively the aspect thereof that bindscorresponding ligand-binding entities. The ligand moiety need notcomprise, and the term generally does not denote, structural featuresother than those required for ligand binding.

“mEq” herein means milliequivalent(s).

“μEq” herein means microequivalent(s).

“Mimetic(s)” herein means a chemical entity with structural orfunctional characteristics of another, generally unrelated chemicalentity. For instance, one kind of hormone mimetic is a non-peptideorganic molecule that binds to the corresponding receptor in the sameway as the corresponding hormone.

“mM” means millimolar; 10⁻³ moles per liter.

“Modified protein(s),” “modified polypeptide(s),” or “modifiedfragment(s)” herein means a protein or a polypeptide or a fragment of aprotein or polypeptide comprising a chemical moiety (structure) otherthan those of the twenty naturally occurring amino acids that formnaturally occurring proteins. Modifications most often are covalentlyattached, but can also be attached non-covalently to a protein or otherpolypeptide, such as a fragment of a protein.

“Moiety(s)” herein means a molecular entity that embodies a specificstructure and/or function, without extraneous components. For instance,in most cases, only a small part of a ligand-binding protein isresponsible for ligand binding. This part of the protein, whethercontinuously encoded or discontinuously, is an example of aligand-binding moiety.

“Naturally occurring” means occurs in nature, without humanintervention.

“Non-naturally occurring” means does not occur in nature or, if itoccurs in nature, is not in its naturally occurring state, environment,circumstances, or the like.

“PBS” means phosphate buffered saline.

“Peptibody” refers to a molecule comprising an antibody Fc domain C_(H)2and C_(H)3 antibody domains) that excludes antibody C_(H)1, C_(L),V_(H), and V_(L) domains as well as Fab and F(ab)2, wherein the Fcdomain is attached to one or more peptides, preferably apharmacologically active peptide, particularly preferably a randomlygenerated pharmacologically active peptide. The production ofpeptibodies is generally described in PCT publication WO00/24782,published May 4, 2000, which is herein incorporated by reference in itsentirety, particularly as to the structure, synthesis, properties, anduses of peptibodies.

“Peptide(s)” herein means the same as polypeptide; often, but notnecessarily, it is used in reference to a relatively short polypeptide,

“pH” is used in accordance with its well-known and universal definitionas follows:

pH=−log [H₃O⁺].

“Pharmaceutical” as used herein means is acceptable for use in a humanor non-human subject for the treatment thereof, particularly for use inhumans, and approved therefor by a regulatory authority empowered toregulate the use thereof such as, for example, the Food and DrugAdministration in the United States, European Agency for the Evaluationof Medicinal Products, Japan's Ministry of Health, Labor and Welfare, orother regulatory agency such as those listed in R. Ng, DRUGS: FROMDISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, N.J.) (2004), which isherein incorporated by reference in its entirety, particularly as toregulatory authorities concerned with drug approval, especially aslisted in Chapter 7. As used herein the phrase “wherein the compositionhas been approved for pharmaceutical use by an authority legallyempowered to grant such approval” means an entity or institution or thelike, established by law and by law charged with the responsibility andpower to regulate and approve the use of drugs for use in humans, and insome cases, in non-humans. Approval by any one such agency anywheremeets this qualification. It is not necessary for the approving agencyto be that of the state in witch, for instance, infringement isoccurring. Example of such entities include the U.S Food and DrugAdministration and the other agencies listed herein above.

As used herein, “pharmaceutical” also may refer to a product produced inaccordance with good manufacturing practices, such as those describedin, among others, Chapter 9 and Chapter 10, of R. Ng, DRUGS: FROMDISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, N.J.) (2004), which isherein incorporated by reference in its entirety, particularly in partspertinent to good manufacturing practices for pharmaceutical proteinformulations, in particular, as set forth in Chapters 9 and 10.

“Pharmaceutically acceptable” is used herein in accordance with itswell-known meaning in the art to denote that which is acceptable formedical or veterinary use, preferably for medical use in humans,particularly approved for such use by the US Food and DrugAdministration or other authority as described above regarding themeaning of “pharmaceutical.”

“Polypeptide(s)” see “Protein(s).”

“Precursor(s)” is used herein in accordance with its well-known meaningin the art to denote an entity from which another entity is derived. Forinstance, a precursor protein is a protein that undergoes processing,such as proteolytic cleavage or modification, thereby giving rise toanother precursor protein (which will undergo further processing) or amature protein.

“Protein(s)” herein means a polypeptide or a complex of polypeptides, inaccordance with its well-known meaning in the art. As used herein,“protein(s)” includes both straight chain and branched polypeptides. Itincludes unmodified and modified polypeptides, including naturallyoccurring modifications and those that do not occur naturally. Suchmodifications include chemical modifications of the termini, the peptidebackbone, and the amino acid side chains; amino acid substitutions,deletions and additions; and incorporation of unusual amino acids andother moieties, to name just a few such modifications. It also includes“engineered” polypeptides and complexes thereof, such as, but notlimited to, any polypeptide or complex of polypeptides that has beendeliberatively altered in its structure by, for instance, recombinantDNA techniques, chemical synthesis, and/or covalent modification,including deliberate alteration of amino acid sequence and/orposttranslational modifications.

“Protonation” means the addition of at least one hydrogen.

“Self-buffering” means the capacity of a substance, such as apharmaceutical protein, to resist change in sufficient for a givenapplication, in the absence of other buffers.

“Semi-de novo” herein means (a) partly designed in accordance with aparticular reference and or produced from a precursor, and (b) partlydesigned without reference to a particular reference (such as designedsolely by general principles and not based on any particular reference).For example, a polypeptide made by producing a first peptide in abacterial expression system, producing a second peptide by chemicalsynthesis, and then joining the two peptides together to form thepolypeptide.

“Semi-synthesis” means as used herein a combination of chemical andnon-chemical methods of synthesis.

“Subject” means a vertebrate, such as a mammal, such as a human. Mammalsinclude, but are not limited to, humans, farm animals, sport animals,and pets. Subjects in need of treatment by methods and/or compositionsof the present invention include those suffering from a disorder,dysfunction, or disease, or a side effect thereof, or from a side effectof a treatment thereof.

“Substantially” is used herein in accordance with its plain and ordinarydefinition to mean to a great extent or degree. For example,substantially complete means complete to a great extent, complete to agreat degree. By way of further illustration, substantially free ofresidue means to a great extent free of residue, free of residue to agreat degree. Should numerical accuracy be required, depending oncontext, “substantially,” as used herein means, at least, 80% or more,particularly 90% or more, very particularly 95% or more.

“Therapeutically effective” is used herein in accordance with itswell-known meaning in the art to denote that which achieves animprovement in the prognosis or condition of a subject or that otherwiseachieves a therapeutic objective, including, for instance, a reductionin the rate of progress of a disease even if a subject's condition,nonetheless, continues to deteriorate.

“Therapeutically effective amount” generally is used to qualify theamount of an agent to encompass those amounts that achieve animprovement in disorder severity. For example, effective neoplastictherapeutic agents prolong the survivability of the subject, inhibit therapidly-proliferating cell growth associated with the neoplasm, oreffect a regression of the neoplasm. Treatments that are therapeuticallyeffective within the meaning of the term as used herein, includetreatments that improve a subject's quality of life even if they do notimprove the disease outcome per se.

“Treat,” “treating,” or “treatment” are used broadly in relation to theinvention and each such term encompasses, among others, preventing,ameliorating, inhibiting, or curing a deficiency, dysfunction, disease,or other deleterious process, including those that interfere with and/orresult from a therapy.

“Variant(s)” herein means a naturally occurring or synthetic version of,for instance, a protein that is structurally different from the originalbut related in structure and/or function, such as an allelic variant, aparalog, or a homolog of a protein.

DESCRIPTION OF THE INVENTION

The invention provides for the first time self-buffering proteinformulations, particularly biopharmaceutical protein formulations,methods for making the formulations, and methods for using theformulations, among other things. Any protein that provides sufficientbuffer capacity within the required pH range at a concentration suitablefor its intended use can be prepared as a self-buffering proteinformulation in accordance with the invention. The invention can bepracticed with a variety of proteins, including both naturally-occurringproteins and “engineered” proteins, particularly biopharmaceuticalproteins, as discussed further below.

The utility of proteins, particularly biopharmaceutical proteins, to beformulated in self-buffering compositions, particularly pharmaceuticallyacceptable compositions, has not been recognized prior to the inventionherein disclosed. The influence of proteins in the regulation ofphysiological pH has been recognized and studied for some time. However,it has not heretofore been recognized that proteins, particularlybiopharmaceutical proteins, can have enough buffer capacity to maintaina formulation within a desired pH range, without additional bufferingagents.

Biopharmaceutical proteins for use in the United States are formulatedas buffered solutions, unbuffered solutions, amorphous or crystallinesuspensions, and lyophilates.

Most of the buffered solution formulations use a conventional bufferingagent. Two proteins, Pulmozyme® and Humulin®, are formulated assolutions without conventional buffering agents. Neither of theseproteins provides substantial self-buffering capacity in theformulations.

Pulmozyme® has a molecular weight of about 37,000 Daltons and contains 5histidines, 22 aspartic acids, and 12 glutamic acids, among its 260amino acids. The buffering capacity of the protein within 0.5 pH unitsof pH 6.3 is determined substantially by its histidine content. On thisbasis, the upper limit of the self-buffering capacity of the formulationis determined by the effective concentration of the histidine residues,0.15 mM. The molar concentration of aspartic acid and glutamic acid inthe formation is 0.9 mM. The total molar concentration of all threeamino acids together, thus, is just a little over 1 mM, at theconcentration of the formulation.

Humulin® is formulated at 3.5 g/ml. It has a molecular weight of about6,000 Daltons and contains 2 aspartic acids, 8 glutamic acids, and 2histidines. None of these amino acids is a particularly effective bufferat the pH of the formulation: 7.0 to 7.8. At this concentration themolar concentration of histidines, which are closest in pK_(a) to the pHof the formulation, is 1.16 mM.

The biopharmaceutical lyophilates are reconstituted prior to use formingsolutions or suspensions. Most of the lyophilates contain conventionalbuffers that maintain the proper pH of the reconstituted formulations. Afew others, in which the protein concentration is low or the pH must below (less than 3) or high (greater than 9.5), are, effectivelyunbuffered.

Thus, buffering is achieved in current biopharmaceutical proteinformulations using conventional buffering agents. The ability ofproteins by themselves to buffer pharmaceutical protein formulations hasnot been fully appreciated and has not been used for the manufacture ofprotein pharmaceuticals.

The determination of protein buffer capacity, typically, is important todeveloping self-buffering protein formulations in accordance with theinvention. Pertinent thereto, methods for measuring buffer capacity andfor determining the buffer capacity of proteins are described below. Toallow ready comparability of data, protein buffer capacity must beexpressed in comparable units and/or related to a buffer standard.Accordingly, the following section describes pH metrics and standardsthat meet these requirements, in accordance with the invention.

1. Buffering

A widely accepted definition of buffering is the resistance to change inpH of a composition upon addition of acid or base. Buffer capacity thusoften is defined as the ability of a composition to resist pH change.

Typically buffer capacity is expressed in terms of the amount of strongacid or base required to change the pH of a composition a given amount.Van Slyke provided the most widely used quantitative measure of buffercapacity, according to which, for a solution, buffer capacity isexpressed as the amount of strong acid or base required to change the pHof one liter of the solution by one pH unit under standard conditions oftemperature and pressure.

According to this measure, for instance, the buffer capacity of 1 literof 5 mM HOAc, 5 mM NaOAc, pH 4.76 in pure water is 4.09×10⁻³ moles of aunivalent strong base (i.e., 4.09×10⁻³ equivalents of base), which canbe calculated as fellows.

The Henderson-Hasselbalch equation for the solution is:

pH=log {[5 mM] NaOAc/[5 mM] HOAc}+4.76

Accordingly, the concentration, X, of a univalent strong base requiredto increase the pH of this buffer is:

4.76 to 5.76 is 5.76=log {[5 mM+X mM] NaOAc/[5 mM−X mM] HOAc}+4.76

Thus:

1.00=log {[5 mM+X mM] NaOAc/[5 mM−X mM] HOAc}

10.0=[5 mM+X mM] NaOAc/[5 mM−X mM] HOAc

10.0=(5 mM+X mM)/(5 mM−X mM)

50 mM−10X mM=5 mM+X mM

1X mM=45 mM

X=4.09 mM,

which, for one liter yields:

(4.09×10⁻³ moles/liter)(1 liter)(1 equivalent/mole)=4.09×10⁻³equivalents

Thus, according to this measure, the buffer capacity of 1 liter of a 10mM acetate buffer containing 5 mM NaOAc and 5 mM HOAC at a pH of 4.76 inpure water is 4.09×10⁻³ equivalents of base per liter per pH unit. Putother ways, the buffer capacity of the solution is 4.09 milliequivalentsof base per liter per pH unit, 4.09 microequivalents of base permilliliter per pH unit, 0.409 microequivalents of base per 100microliters per pH unit, 40.9 nanomoles of base per 10 microliters perpH unit, and 4.09 nanonmoles of base per microliter per pH unit.

The same calculation yields the following buffer capacity for otherconcentrations of this acetate buffer at pH 4.76. A 2 mM acetate bufferas above has a buffer capacity of 0.818 mEq per liter per pH unit. At 4mM the buffer capacity is 1.636 mEq per liter per pH unit. The capacityat 5 mM is 2.045 mEq per liter per pH unit. At 7.5 mM the capacity is3.068 mEq per liter per pH unit. At 10 mM the acetate buffer has abuffer capacity of 4.091 mEq per liter per pH unit. At 15 mM itscapacity is 6.136 mEq per liter per pH unit.

It is worth noting that an acetate buffer solution at the pK_(a) ofacetic acid (pH 4.76) is equimolar in acetic acid and acetate base.(i.e., at the pK_(a) the acid and base are present in equal amounts). Asa result, the resistance to change in pH (buffer capacity) of an acetatebuffer at the pK_(a) of acetic acid is the same for addition of acid andbase. The equipoise to acid and base is a general characteristic ofbuffering agents in buffers at a pH equal to their pK_(a).

At any other pH a buffer will contain different amounts of acid and baseforms and, therefore, its resistance to change (i.e., its buffercapacity) upon addition of acid will not be the same as its resistanceto change upon addition of base. As a result, it is preferable to definethe capacity of such buffers in terms of (i) the amount of acid requiredto lower the pH by one unit, and (ii) the amount of base required toraise the pH by one unit.

The partitioning in a buffer between acid and base forms in a givencomposition, such as a pH standard, can be calculated at any pH andbuffer concentration using the procedures set forth above in describingthe buffer capacity of 10 mM NaOAc at pH 4.76 plus or minus (containingequimolar amounts of acetic acid and sodium acetate). And the resultscan be used to define the buffer capacity of a standard for referenceuse.

Thus, for instance, the partitioning of acetic acid into acetic acid andacetate base in a solution at pH 5.0 can be calculated readily using theforegoing procedures, and from this the buffer capacity can becalculated for both base and for acid addition. Calculated this way, thetheoretical buffer capacity of 10 mM sodium acetate buffer over therange from pH 5.0 to 5.5 is approximately 2.1 mM per 0.5 pH unit and 4.2mM per pH unit. Put another way, the buffer capacity of the buffer,theoretically, is approximately 4.2 μEq per ml of buffer solution perunit of pH change. Similarly, the theoretical buffer capacity of 10 mMsodium acetate buffer over the range from pH 5.0 to 4.0 is 4.9 mM, and,put another way, 4.9 μEq per ml of buffer per unit of pH change over agiven range of pH.

While such calculations often are quite useful in many cases, empiricalstandards and empirical determinations are preferred. Among particularlypreferred empirical standards are sodium acetate buffers over the rangeof pH 5.0 to 4.0 and pH 5.0 to 5.5 as exemplified in Examples 1 and 2.Especially preferred are sodium acetate buffers in accordance therewithin which the total acetate concentration is, in particular, 10 mM,preferably 5 mM, especially 4 mM, among others as set forth elsewhereherein.

Acetate buffers at pH 5.0 are more resistant to change in pH uponaddition of acid than upon addition of base, as discussed above. In apreferred empirical standard of buffer capacity, the buffer capacity ofa standard acetate buffer such as these is defined as: (i) the slope ofthe least squares regression line calculated for base titration data forthe buffer from pH 5.0 to pH 5.5, and (ii) the slope of the leastsquares regression line calculated for acid titration data for thebuffer from pH 5.0 to pH 4.0. The preparation of standard acetatebuffers and the determination of their buffer capacities are describedin Examples 1, 2, and 3. It is to be appreciated that much the samemethods can be used to establish and use buffer capacity standards usingother suitable buffering agents.

In measuring the buffer capacity of a self-buffering protein compositionin accordance with the invention, it often is convenient to express thebuffer capacity in terms of the concentration of a standard buffer atthe same pH having the same buffer capacity. When a standard is usedthat is not at the pK_(a) of the buffering agent, such as a sodiumacetate buffer initially at pH 5.0, in accordance with the invention theself-buffering composition is defined as having a buffer capacity equalto or greater than that of the standard, if either its buffer capacityupon base titration or its buffer capacity upon acid titration (or both)is equal to or exceeds the corresponding buffer capacity of thestandard.

It is to be further appreciated that the pH of self-buffering proteincompositions in accordance with the invention generally will not be atthe pK_(a) of the self-buffering protein, or any acid-base substituenttherein. Indeed proteins are polyprotic and, as discussed herein, oftenwill have several substituents, each with a somewhat different pK_(a)that contribute to its buffer capacity in a given pH range. Accordingly,the buffer capacity of self-buffering protein formulations in accordancewith the invention preferably is determined empirically by both acidtitration and base titration over a given range of pH change from thedesired pH of the composition. In preferred embodiments in this regard,the buffer capacity is determined by titrating with acid and separatelywith base over a change of respectively + and −1 pH unit from thestarting pH of the formulation. In particularly preferred embodiments,the titration data is collected for a change in pH of plus or minus 0.5pH units. As described in the Examples, the buffer capacity is the slopeof the least squares regression line for the data for pH as a functionof equivalents of acid or base added to the composition over the rangeof titration.

a. Empirical Measures and Standards of Buffer Capacity

In certain preferred embodiments of the invention, the measure of buffercapacity is an empirical standard. Among preferred empirical standardsin this regard are a particular volume of an aqueous solution at aparticular temperature and a particular pH, containing a particularbuffering agent at a particular concentration and either no othercomponents than water, or one or more other particular components, eachat a particular concentration.

A particularly preferred specific standard for determining buffercapacity in accordance with various aspects and preferred embodiments ofthe invention is 10 mM sodium acetate pH 5.00 in pure water free ofother constituents at 21° C. in equilibrium with ambient air at 1atmosphere, as described in Examples 1 and 2, preferably expressed inequivalents per unit volume per pH unit, such as μEq/ml-pH. Buffercapacity of the standard should be measured empirically as described inExamples 1, 2, and 3, and as further discussed elsewhere herein.

A particularly preferred specific standard for determining buffercapacity in accordance with various aspects and preferred embodiments ofthe invention is 10 mM sodium acetate pH 4.76 in pure water free ofother constituents at 21° C. in equilibrium with ambient air at 1atmosphere, as described in Examples 1 and 2, preferably expressed inequivalents per unit volume per pH unit, such as μEq/ml-pH. Buffercapacity of the standard should be measured empirically as described inExamples 1, 2, and 3, and as further discussed elsewhere herein.According to the Henderson-Hasselbalch equation, as noted above, thecalculated buffer capacity of this standard over the range of pH 4.76plus or minus 1 pH unit is 4.09 microequivalents per milliliter per pHunit (4.09 μEq/ml-pH).

A variety of other buffers are available for use as standards in otherranges of pH in accordance with various aspects and preferredembodiments of the invention in this regard. Reference buffers areparticularly preferred in this regard, such as those well-known androutinely employed for analytical chemistry determinations. A variety ofsuch buffering agents are set forth in textbooks on analytical chemistryand in monographs on the accurate determination of pH and buffercapacity.

Also useful in the invention in this regard are biological buffers, suchas those described in, among other texts: TEITZ TEXTBOOK OF CLINICALCHEMISTRY, 3^(rd) Ed., Burtis and Ashwood, eds., W.B. Saunders Company,Philadelphia, Pa. (1999), in particular in Tables 50-13 to 50-16, whichare herein incorporated by reference in their entireties as to bufferingagents and buffers and their use as pH and/or buffer capacity standardsin accordance with the invention in this respect; THE TOOLS OFBIOCHEMISTRY, Terrance G. Cooper, John Wiley & Sons, New York, N.Y.(1977), in particular Chapter 1, pages 1-35, which is hereinincorporated by reference in its entirety as to buffering agents andbuffers and their use as pH and buffer capacity standards in accordancewith the invention in this respect, most particularly as to Tables 1-3,1-4, and 1-5 and text relating thereto, and PROTEIN PURIFICATIONPRINCIPLES AND PRACTICE, 3^(rd) Ed., Robert K. Scopes, Springer-Verlag,New York, N.Y. (1994), in particular pages 160-164, especially thereinTables 6.4 and 6.5 and text relating thereto, Chapter 12, section 3,pages 324-333, especially therein Tables 12-4 and 12-5 and text relatingthereto, and all of Appendix C: Buffers for Use in Protein Chemistry,which are herein incorporated by reference in their entirety as tobuffering agents and buffers and their use in accordance with theinvention in this respect.

Since some dissolved gases in water react with OH⁻ and/or H₃O⁺, however,the empirically determined buffer capacity of the standard solution mayvary somewhat from the theoretical value. Hence, the definition of thestandard requires that the solution be in equilibrium with theatmosphere at a pressure of 1 atmosphere. In addition, the bufferstandard must be held in and contacted only with materials that do notalter its components or its buffer capacity, such as those that leachacids, bases, or other reactants that may alter the effectiveconcentration or activity of the acetate buffer in any way that wouldalter its buffer capacity. Given both of the foregoing, atmosphericequilibration and inertness of the container, buffer capacity of thestandard will scale directly and linearly with its volume. Accordingly,the buffer capacity of 100 ml will be 1/10 that of 1.00 liter, and thebuffer capacity of 10 ml will be 1/100 that of 1.00 liter. Accordingly,the volume of the standard can be adjusted for convenience and thennormalized back to 1 liter as desired.

It may net always be convenient to make the foregoing 10 mM acetatebuffer capacity standard for field use. However, a variety of otherbuffer capacity standards can be made and used in the same way as theacetate buffer, using a variety of other buffering agents. Provided onlythat the buffering standards are prepared properly, they can becalibrated against the acetate buffering standard described above andthen used in the field. The results obtained with such alternativestandards may then be expressed in terms of the foregoing acetatestandard without substantial distortion or error.

The buffer capacity of such alternative standards also can be calibratedby calculation. To do so, the buffer capacity of the alterative standardis determined directly and expressed in mEq per unit volume per unit ofpH. Determinations based on the alternative standard then can benormalized to the acetate standard using the ratio between the bufferingcapacities expressed in mEq per unit volume per unit of pH of thealternative and the acetate standards.

Using such methods, which are commonly employed in metrology to relatefield standards back to a reference standard, the acetate bufferstandard described above provides a portable, scalable, reliable, andaccurate reference for determining the buffer capacity of anycomposition that readily can be compared with disparate measures made onother compositions using similar methods.

b. Preparation of Buffer Capacity Standards

Buffer capacity standards can be prepared using well-established methodsof analytical chemistry. See for instance, ANALYTICAL CHEMISTRY, 3^(rd)Ed., Douglas A. Skoog and Donald M. West, Holt, Rinehart and Winston,New York (1979), particularly chapter 9 (pages 186-226), chapter 10(pages 227-233), and methods described on pages 553-588; TEITZ TEXTBOOKOF CLINICAL CHEMISTRY, 3^(rd) Ed., Burtis and Ashwood, eds., W.B.Saunders Company, Philadelphia, Pa. (1999), in particular Chapter 1regarding general laboratory techniques for preparing and calibratingbuffers and Tables 50-13 to 50-16; THE TOOLS OF BIOCHEMISTRY, TerranceG. Cooper, John Wiley & Sons, New York, N.Y. (1977), in particularChapter 1, pages 1-35, and Tables 1-3, 1-4, and 1-5 and text relatingthereto; PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, 3^(rd) Ed.,Robert K. Scopes, Springer-Verlag, New York, N.Y. (1994), in particularpages 160-164, especially therein Tables 6.4 and 6.5 and text relatingthereto, Chapter 12, section 3, pages 324-333, especially therein Tables12-4 and 12-5 and text relating thereto, and all of Appendix C: Buffersfor Use in Protein Chemistry; and REMINGTON: THE SCIENCE AND PRACTICE OFPHARMACY, 21^(st) Ed., Beringer et at. Editors, Lippincott, Williams &Wilkins, Philadelphia, Pa. (2005), particularly in parts relating tobuffering agents, buffers, buffer capacity and the like; each of whichis herein incorporated by reference in its entirety particularly as tothe preparation and use of buffers and buffer capacity standards inaccordance with the invention in this respect.

The water used for preparing buffer capacity standards should be highlypurified, preferably Type I water, such as milliQ water, or tripledistilled water. The buffer reagents should be pure and, in particular,free of any substance that can alter the pH or buffer capacity of thestandard solution, such as Reference Grade or ACS Reagent Grade reagentssuitable for use in demanding analytic chemical analyses, as describedin the foregoing references, TEITZ and REMINGTON cited above inparticular, which are hereby incorporated by reference in theirentireties particularly in parts pertinent to analytical grade water andreagents.

The exact compositions of the buffer reagents must be well established.The molecular weight of the buffer reagents must be known accurately foreach buffer reagent. The molecular weights must be for the exact reagentthat will be used and must include the weight of adducts such ashydrates that are present in the reagent. The effective number ofhydrogen donors or hydrogen acceptors per molecule must be knownaccurately for each buffer reagent. The proportional distribution ofdifferent forms, such as hydrates, must be known for each reagent thatcontains a mixture of such forms. Concentrations of liquid bufferreagents much be known exactly, preferably in moles/volume and inmoles/mass (e.g., moles/liter and moles/gm or kg. Hygroscopic agentsmust be dried to remove moisture so that reagent can be accuratelyweighed.

Generally speaking, the information provided by well-established vendorsof reagents and reference grade chemicals is sufficiently accurate forthe preparation of buffer capacity standards as described above. Andwell-known standard techniques routinely employed in analyticalchemistry can be used to dry “hygroscopic reagents” so that they can beweighed accurately.

As described therein, well established and routinely employed analyticalchemistry methods can be employed to prepare and calibrate acid and basesolutions, such as 1 N HCl and 1 N NaOH (to name just two) for titratingbuffer capacity standard solutions, as well as sample protein solutions,to determine buffer capacity. It should be noted that the preparation ofNaOH solutions for titration should be done so as to eliminateinaccuracies that arise from the interaction of certain dissolved gaseswith basic solutions, and the pH altering effects of their solvation.See for instance Skoog and West (1979) and other references cited aboveregarding the preparation and calibration of buffers and bufferstandards, which are herein incorporated by reference in theirentireties particularly in parts pertinent to the preparation ofstandard solutions for titration, as discussed above.

c. Empirical Measurement of Buffer Capacity

Titration of standards and samples to determine buffer capacity can bedone using well-known, routine methods. Titrations can be carried outmanually. They also can be carried out using an autotitrator. A widevariety of autotitrators that are suitable for use in the invention inthis regard are commercially available from numerous vendors. Methodssuitable for use in the invention in this regard are the same as thosedescribed in the references cited above regarding preparation andcalibration of buffer standards, each of which is incorporated herein byreference in its entirety particularly in parts pertinent to thetitration of known and unknown solutions to determine their buffercapacity.

2. Buffering by Proteins and Protein Buffer Capacity

a. Determination of Protein Hydrogen Equilibria and Buffer Capacity

Proteins invariably contain many acidic and basic constituents. As aresult hydrogen ion equilibrium of proteins is highly complex. In fact,a complete description of the hydrogen ion equilibria of a protein in agiven environment is beyond the reach of current theoretical andcomputational methods. Empirical measurements of protein buffercapacities, thus are preferred. Methods developed for precise empiricalmeasurement of protein hydrogen equilibria, which can be and areroutinely employed by those skilled in the art, are well-suited tomeasuring the buffering properties of proteins pertinent to thedevelopment of self-buffering protein formulations in accordance withthe invention. Thus, the pH titration curves of proteins can bedetermined in accordance with the invention by well-known methods suchas those described in and exemplified by pH titration studies of Tanfordand co-workers on ribonuclease. See C. Tanford, “Hydrogen Ion TitrationCurves of Proteins,” in T. Shedlovsky (ed.), ELECTROCHEMISTRY IN BIOLOGYAND MEDICINE, John Wiley and Sons, New York, 1955, Ch. 13; C. Lanfordand J. D. Hauenstein, J. Am. Chem. Soc. 78, 5287 (1956), C. Tanford,PHYSICAL CHEMISTRY OF MACROMOLECULES, John Wiley and Sons, New York,1961, particularly pages 554-567, all of which are herein incorporatedby reference particularly in parts pertinent to hydrogen ion titrationof proteins and to the determination of buffering action and buffercapacity of proteins.

However, the present invention does not require such precisedeterminations as those described in the foregoing references. Rather,the buffering properties and buffer capacity of proteins in accordancewith the invention can be determined using the methods described instandard references on analytical chemistry and biochemistry, such as,for instance, Skoog (1979), Cooper (1977), and Scopes (1994), citedabove, each of which is herein incorporated by reference in its entiretyparticularly as to the empirical determination of titration curves,particularly of proteins within a given range of pH in accordance withthe invention.

The determination of titration curves and buffer capacity in accordancewith the invention is described in detail for numerous acetate buffersand a variety of pharmaceutical proteins in the Examples below. Thus,the pH titration curves of proteins can be determined empirically inaccordance with such methods as described in the foregoing referencesover particular limited ranges of pH that are of interest to a givenformulation. In many respects these methods are the same as those usedin analytical chemistry for the titration of small molecules such asacetate buffers (as illustrated in the Examples). Somewhat greater caremust be taken, however, in handling proteins to maintain theconformation and function required for effective formulation.

Protein titrations may be carried out manually or using automatedtitrators. Equipment for manual titration and automated titrators arereadily available from a large number of suppliers and vendors. Methodssuitable for determining pH titration curves and buffer capacity ofproteins are exemplified in the Examples by reference to titration ofacetate buffer standards and to titration of several differenttherapeutic proteins over defined ranges of pH. These methods can beemployed to determine the hydrogen ionization behavior and buffercapacity of any other protein in accordance with the invention.

It is a particular aspect of the invention to determine the buffercapacity of proteins as a function of concentration in solution. In apreferred method in this regard, solutions of a given protein areprepared in a graded series of concentrations. A pH titration curve isdetermined for the protein at each concentration over the pH range ofinterest. Preferably titration curves are determined for the range ofinterest using both base titration and acid titration. The data are, incertain preferred embodiments, plotted on a graph of equivalents of acidor base added versus the measured pH of each solution. Typically, forranges of about 0.5 to 1.0 pH unit, the titration data for eachconcentration closely fit a straight line, preferably determined by aleast squares regression analysis. In preferred embodiments in thisregard, buffer capacity for the protein at each concentration is equatedto the slope of the regression line, expressed in units of equivalentsper ml per pH unit (or fractions thereof). Also useful in the inventionin this regard is the relationship between the buffer capacity of theprotein and its concentration. In certain preferred embodiments, thisrelationship is determined by a least squares regression analysis of thebest straight line fit of the buffer capacity data determined inaccordance with the foregoing plotted on a graph of buffer capacityversus protein concentration.

Empirical data on the buffer capacity of proteins in accordance with theinvention preferably is related to the buffer capacity of a standardacetate buffer. That is, in particularly preferred embodiments of theinvention in this regard, the buffer capacity of a given protein at agiven concentration in a given formulation, determined as above, isequated to the concentration of a standard acetate buffer having thesame buffer capacity.

While empirical determinations as described herein are generally acrucial aspect of formulating self-buffering compositions in accordancewith various aspects and preferred embodiments of the invention,theoretical and computational methods also can be productively employedto guide the design, manufacture, and use of such compositions (inconjunction with empirical determinations), as described below.

b. Prediction of Protein Hydrogen Ion Equilibria and Buffer Capacity

The ionization of hydrogen in proteins is complex but can be broken downin general terms into pH ranges defined by the ionizable hydrogens ofamino acid side chains, and the terminal amino and carboxyl groups. ThepK_(a) of terminal carboxyls in polypeptides typically ranges around3.1. The pK_(a) of the acidic hydrogens in the side chains of asparticacid and glutamic acid range around 4.4. The pK_(a) of histidine inpolypeptides ranges around 6.0. The terminal amino group hydrogenionization pK_(a) typically ranges around 7.5. The sulfhydryl incysteine has a pK_(a) range around 8.5. The tyrosine hydroxyl and thelysine amine both have pK_(a)s ranging around 10. The pK_(a) of arginineranges around 12.

Conformational folding typically partitions large polypeptides andproteins in polar solvents into exposed solvent-accessible regions andmore or less non-polar core regions that have little or no contact withthe ambient environment. Folding produces many environments betweenthese two extremes. Furthermore, the micro environment around a givenamino acid side chain in a protein typically is affected by one or moreof: solvent effects; binding of ions; chelation; complexation;association with co-factors; and post-translational modifications; toname just a few possibilities. Each of these can influence the pK_(a) ofa given amino acid ionization in a protein. The pK_(a)s for specificresidues in a given protein, thus, can vary dramatically from that of afree amino acid.

Indeed, the perturbation of pK_(a)s by microenvironments of amino acidsin proteins has been used to study the folding of proteins and thedisposition and charge state of specific amino acids in folded proteins.The protein titration curves reported by Tanford and others are complexwith a few broad features in common. Typically only some of theionizable protons are accounted for in the titration curves. Othersapparently are located in the core and are inaccessible to solvent. ThepK_(a)s of individual side chains of the same type that can be detectedin some cases can be distinguished from one another. Nonetheless, whiledetectably different, their pK_(a)s generally are close to that of thefree amino acid.

The strongest buffering action of proteins does not generally occur atthe isoelectric point, as may be mistakenly supposed. In fact, bufferingdepends on the amino acid side chain hydrogens and the terminalhydrogens, and therefore occurs in ranges spanning the pK_(a)s of theionizable hydrogens in the free amino acids, as discussed above. Themost important of these, for formulating compositions of proteins,especially certain pharmaceutical proteins that are more soluble and/ormore stable, among other things, at weakly acidic pH (pH 4 to 6), isbuffering action that occurs in the range of the pK_(a)s of the carboxylhydrogen of the amino acids aspartic acid and glutamic acid; that is, pH4.0 to 5.5, particularly around 4.5.

There are a variety of ways available for estimating the buffer capacityof a given protein in a given solution at a given pH. Methods range fromhighly technical and complex computer models to those that can becarried out on a hand calculator. None of the methods is complete orentirely accurate; but, they can in some instances provide usefulestimates.

For instance, a potentially useful idea of buffer capacity in someinstances may be calculated for a protein in a solution based on itsamino acid composition, the pK_(a)s (in the solvent in question) of theterminal amine and carboxy groups and the amino acid side hydrogendonors and acceptors, the concentration of the protein, and the pH ofthe solution.

For example, a potentially useful estimate of the buffer capacity of aprotein at pH in the range of the pK_(a) of the side chain carboxylhydrogen of glutamic acid (as a free amino acid), can be gained from themolecular weight of the protein and the number of glutamic acid residuesit contains. Dividing the former by the latter provides the weight perequivalent of glutamic acid and, therefore, the weight per equivalent ofionizable hydrogen at the pK_(a) of glutamic acid. Since glutamic acidand aspartic acid side Chain carboxyl groups have nearly the samepK_(a)s, results of such calculations for the two should be addedtogether to yield an estimate of buffer capacity in a range around boththeir pK_(a)s. The estimated buffer capacity of a solution of theprotein at the pK_(a) can be calculated from the protein's concentrationin the solution and the intrinsic factor just provided, namely weightper equivalent of ionizable hydrogen. Dividing the concentration by theweight per equivalent yields an estimate for the buffer capacity inunits of Eq/volume. Such estimates often will be too high, since someresidues usually are sequestered in regions of the protein notaccessible to the solvent, and, therefore, do not contribute to itsactual buffer capacity. It may be possible in certain instances toaccount for the effect of sequestering on buffer capacity. For instance,a fractional co-efficient that reflects theoretical or empiricalestimates of sequestering can be applied to adjust the originalcalculation.

Such calculations generally will be of less utility and less accuratethan empirical determinations of protein buffer capacity, in accordancewith the methods described elsewhere herein. But they can be useful toprovide rough maximum estimates of the buffer capacity of proteins insolution.

3. Proteins

The invention herein disclosed may be practiced with any protein thatprovides sufficient buffer capacity in a desired pH range within theparameters of protein concentration and the like required for a desiredformulation. Among preferred proteins in this regard are pharmaceuticalproteins for veterinary and/or human therapeutic use, particularlyproteins for human therapeutic use. Also among preferred proteins areproteins that are soluble in aqueous solutions, particularly those thatare soluble at relatively high concentrations and those that are stablefor long periods of time. Additionally, among preferred proteins arethose that have a relatively high number of solvent accessible aminoacids with side chain hydrogen ionization constants near the pH of thedesired buffering action.

Further among preferred proteins of the invention are proteins forpharmaceutical formulations that do not induce a highly deleteriousantigenic response following administration to a subject. Preferred inthis regard are proteins for veterinary and/or human medical use,particularly, regarding the latter, humanized and human proteins.

Further among preferred proteins of the invention are proteins that bindselectively to specific targets, including ligand-binding proteins andprotein ligands. Antigen-binding proteins, proteins derived therefrom,and proteins related thereto are among the particularly preferredembodiments of the invention in this regard. Highly preferred proteinsof the invention in this regard are antibodies and proteins derived fromantibodies or incorporating antibodies, in whole or part, including, toname just a few such entities: monoclonal antibodies, polyclonalantibodies, genetically engineered antibodies, hybrid antibodies,bi-specific antibodies, single chain antibodies, genetically alteredantibodies, including antibodies with one or more amino acidsubstitutions, additions, and/or deletions (antibody muteins), chimericantibodies, antibody derivatives, antibody fragments, which may be fromany of the foregoing and also may be similarly engineered or modifiedderivatives thereof, fusion proteins comprising an antibody or a moietyderived from an antibody or from an antibody fragment, which may be anyof the foregoing or a modification or derivative thereof, conjugatescomprising an antibody or a moiety derived from an antibody, includingany of the foregoing, or modifications or derivatives thereof, andchemically modified antibodies, antibody fragments, antibody fusionproteins, and the like, including all of the foregoing.

a. Antibodies, Antibody-Derived and Antibody-Related Proteins and theLike

Among particularly preferred proteins in accordance with the inventionare antibody polypeptides, such as heavy and light chain polypeptidesthat have the same amino acid sequence as those that occur in and makeup naturally-occurring antibodies, such as those that occur in sera andantisera, including such polypeptides and proteins isolated from naturalsources, as well as those that are made by hybridoma technologies, byactivation of an endogenous gene (by homologous or non-homologousrecombination, for instance), by expression of an exogenous gene underthe control of an endogenous transcription control region, by expressionof an exogenous expression construct, by semi-synthesis and by de novosynthesis, to name some techniques commonly employed for makingantibodies and antibody-related polypeptides and proteins that can beused to produce antibody polypeptides and proteins in accordance withthe invention.

Included among these antibody-related polypeptides and proteins arethose in whole or part having a de novo amino acid sequence, those thatcomprise all or one or more parts of an antibody (that is: a continuouschain of amino acids having the same sequence as any four or moreresidues in the amino acid sequence of a naturally occurring antibodypolypeptide), those having an amino acid sequence that matches in someway that of a naturally occurring antibody, but differs from it in otherways, those that have the same but different amino acid sequences as anaturally occurring counterpart or sequence relating thereto, but differfrom the counterpart in one or more post-translational modifications,and those comprised in part of any of the foregoing (in part or inwhole) fused to one or more polypeptide regions that can be of orderived from or related to a second, different antibody polypeptide, andcan be of or derived from any other polypeptide or protein, whethernaturally occurring, resembling but differing therefrom, having asemi-de novo amino acid sequence and/or a de novo sequence, amongothers. Such hybrids are generally referred to herein as fusionpolypeptides and/or fusion proteins.

Further among preferred proteins in accordance with the invention hereindescribed are modified proteins in accordance with all of the foregoing.Included among such modified proteins are proteins modified chemicallyby a non-covalent bond, covalent bond, or both a covalent andnon-covalent bond. Also included are all of the foregoing furthercomprising one or more post-translational modifications which may bemade by cellular modification systems or modifications introduced exvivo by enzymatic and/or chemical methods, or introduced in other ways.

Among preferred proteins of the invention in this regard are Fabfragment(s), such as those produced by cleaving a typical dimeric (LH)₂antibody with certain protease that leave the light chain intact whilecleaving the heavy chains between the variable region and the adjacentconstant region, “above” the disulfide bonds that hold the heavy chainstogether. Such cleavage releases one Fe fragment comprising theremaining portions of the heavy chains linked together, and two dimericFab fragments each comprising an intact light chain and the variableregion of the heavy chain. Fab fragments also can be produced by othertechniques that do not require isolation of a naturally occurringantibody and/or cleavage with a protease.

Also preferred are Fab₂ fragment(s) such as those produced in much thesame manner as Fab fragments using a protease that cleaves “between orbelow” the disulfide bonds. As a result, the two Fab fragments are heldtogether by disulfide bonds and released as a single Fab₂ fragment. Fab₂fragments can be produced by many other techniques including those thatdo not require isolation of an intact antibody or cleavage with aprotease having the required specificity. Furthermore, both mono- andbi-specific Fab₂ fragments can now be made by a variety of routinetechniques.

Also among preferred proteins in this regard are Fab₃ fragments, whichare engineered antibody fragments in which three Fab fragments arelinked together. Fab₃ fragments can be mono-, bi-, or tri-specific. Theycan be made in a variety of ways well-known to those of skill in thepertinent arts.

Among other preferred proteins in this regard are Fc fragments(s), suchas those produced by cleavage with a protease in the same manner usedfor the production of either Fab fragments or Fab₂ fragments. However,for the production of Fc fragments, the dimeric heavy chain containingfragments are isolated rather than the light chain containing fragments.Fc fragments lack antigen combining sites, but comprise effector regionsthat play a role in physiological processes involving antibodies. Fcfragments can be made by a variety of techniques that are well-known androutinely employed by those of skill in the art for this purpose.

Among other preferred proteins in this regard are single-chain variablefragments (“scFv(s)”), scFv(s) are fusion proteins made by joining thevariable regions of the heavy and light chains of an immunoglobulin. Theheavy and light chains in an scFv typically are joined by a shortserine, glycine linker. scFv(s) have the same specificity as theantibodies from which they were derived. Originally produced throughphage display, scFv(s) now can be made by a variety of well-knownmethods.

Also preferred are Bis-scFv(s) which are fusions of two scFv(s).Bis-scFv(s) can be mono- or bi-specific. A variety of methods arewell-known and can be applied in making Bis-scFv(s) in accordance withthe invention.

Also preferred in accordance with the invention in this regard areminibodies; mono- and bi-specific diabodies; mono-, bi-, andtri-specific triabodies; mono-, bi-, tri-, and tetra-specifictetrabodies; VhH domains; V-NAR domains; V_(H) domains; V_(L) domains;camel Igs; Ig NARs; and others.

Also among preferred embodiments in accordance with various aspects andpreferred embodiments of the invention in these and other regards areproteins comprising one or more CDR and/or CDR-derived and/orCDR-related regions of an antibody or one or more FR and/or FR-derivedand/or FR-related regions of an antibody. In this regard CDR meanscomplementary determining region; that is, a hypervariable region of alight or heavy chain of an antibody, typically about 9 to 12 amino acidsin length that usually is an important part of an antigen specificbinding moiety of an antibody. FR in this regard means a frameworkregion of an antibody; that is, a region of about 15 to 20 amino acidsthat separates CDRs in the antigen specific binding moiety of anantibody. The terms CDR-derived and CDR-related, and the termsFR-derived and FR-related have the same meanings as to CDR and FR,respectively, as set forth in the above Glossary for the termsantibody-derived and antibody-related as to the term antibody.

Regarding antibodies, antibody-derived, and antibody-related proteins inaccordance with the foregoing and with other aspects of the inventionherein disclosed, see, for instance, Protein Engineering: Principles andPractice, Jeffrey L. Cleland and Chares S. Craik, eds. Wiley-Liss, Inc.,New York (1996), particularly therein Kelley, Robert. F., “EngineeringTherapeutic Antibodies,” Chapter 15, pp. 399-434 and Hollinger. P. &Hudson, P., “Engineered antibody fragments and the rise of singledomains,” Nature Biotechnology, September 2005, 1126-1136, each of whichis herein incorporated by reference in its entirety particularly inparts pertinent to the structure and engineering of antibodies,particularly biopharmaceutical antibodies, and antibody-derived andantibody-related proteins, particularly antibody-derived andantibody-related pharmaceutical proteins in accordance with theinvention herein described.

As to all of the foregoing, particularly preferred in the invention arehuman, humanized, and other proteins that do not engender asignificantly deleterious immune responses when administered to a human.Also preferred in the invention are proteins in accordance with all theforegoing that similarly do not cause a significantly deleterious immuneresponses on administration to non-humans.

Among very particularly preferred proteins in accordance with theinvention in these regards are fusion proteins comprising antibodiesand/or antibody-derived proteins, polypeptides, or fragments or thelike, including all of those described above.

Among very particularly preferred fusion proteins of the invention inthis regard are fusion proteins comprising an antibody orantibody-derived protein or fragment such as those described above and aligand-binding moiety, such as those illustratively described herein.

b. Target Binding Proteins

Also among preferred proteins of the invention in this regard areantibodies and other types of target binding proteins, and proteinsrelating thereto or derived therefrom, and protein ligands, and proteinsderived therefrom or relating thereto. Among especially preferredligand-binding proteins in this regard are proteins that bind signal andeffector proteins, and proteins relating thereto or derived therefrom.

Among such binding proteins, including antibodies, including proteinsderived therefrom and proteins related thereto, are those that bind toone or more of the following, alone or in any combination:

-   -   (i) CD proteins including but not limited to CD3, CD4, CD8,        CD19, CD20, and CD34;    -   (ii) HER receptor family proteins, including, for instance,        HER2, HER3, HER4, and the EGF receptor;    -   (iii) cell adhesion molecules, for example, LFA-1, Mol, p        150,95, VLA-4, ICAM-1, VCAM, and alpha v/beta 3 integrin;    -   (iv) growth factors, including but not limited to, for example,        vascular endothelial growth factor (“VEGF”); growth hormone,        thyroid stimulating hormone, follicle stimulating hormone,        luteinizing hormone, growth hormone releasing factor,        parathyroid hormone, mullerian-inhibiting substance, human        macrophage inflammatory protein (MIP-1-alpha), erythropoietin        (EPO), nerve growth factor, such as NGF-beta, platelet-derived        growth factor (PDGF), fibroblast growth factors, including, for        instance, aFGF and bFGF, epidermal growth factor (EGF),        transforming growth factors (TGF), including, among others,        TGF-alpha and TGF-beta, including TGF-beta1, TGF-beta2,        TGF-beta3, TGF-beta4, or TGF-beta5, insulin-like growth        factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain        IGF-I), and osteoinductive factors;    -   (v) insulins and insulin-related proteins, including but not        limited to insulin, insulin A-chain, insulin B-chain,        proinsulin, and insulin-like growth factor binding proteins;    -   (vi) coagulation and coagulation-related proteins, such as,        among others, factor VIII, tissue factor, von Willebrands        factor, protein C, alpha-1-antitrypsin, plasminogen activators,        such as urokinase and tissue plasminogen activator (“t-PA”),        bombazine, thrombin, and thrombopoietin;    -   (vii) colony stimulating factors (CSFs), including the        following, among others, M-CSF, GM-CSF, and G-CSF;    -   (viii) other blood and serum proteins, including but not limited        to albumin, IgE, and blood group antigens;    -   (ix) receptors and receptor-associated proteins, including, for        example, flk2/flt3 receptor, obesity (OB) receptor, growth        hormone receptors, and T-cell receptors;    -   (x) neurotrophic factors, including but not limited to,        bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4,        -5, or -6 (NT-3, NT-4, NT-5, or NT-6);    -   (xi) relaxin A-chain, relaxin B-chain, and prorelaxin;    -   (xii) interferons, including for example, interferon-alpha,        -beta, and gamma;    -   (xiii) interleukins (ILs), e.g., IL-1 to IL-10;    -   (xiv) viral antigens, including but not limited to, an AIDS        envelope viral antigen;    -   (xv) lipoproteins, calcitonin, glucagon, atrial natriuretic        factor, lung surfactant, tumor necrosis factor-alpha and -beta,        enkephalinase, RANTES (regulated on activation normally T-cell        expressed and secreted), mouse gonadotropin-associated peptide,        Dnase, inhibin, and activin;    -   (xvi) integrin, protein A or D, rheumatoid factors,        immunotoxins, bone morphogenetic protein (BMP), superoxide        dismutase, surface membrane proteins, decay accelerating factor        (DAF), AIDS envelope, transport proteins, homing receptors,        addressins, regulatory proteins, immunoadhesins, antibodies; and    -   (xvii) biologically active fragments or variants of any of the        foregoing.

As to all of the foregoing, particularly preferred are those that areeffective therapeutic agents, particularly those that exert atherapeutic effect by binding a target, particularly a target amongthose listed above, including targets derived therefrom, targets relatedthereto, and modifications thereof.

c. Particular Illustrative Proteins

Among particular illustrative proteins are certain antibody andantibody-related proteins, including peptibodies, such as, for instance,those listed immediately below and elsewhere herein:

OPGL specific antibodies and peptibodies and the like (also referred toas RANKL specific antibodies, peptibodies and the like), including fullyhumanized and human OPGL specific antibodies, particularly fullyhumanized monoclonal antibodies, including but not limited to theantibodies described in International Publication Number WO 03/002713,which is incorporated herein in its, entirety as to OPGL specificantibodies and antibody related proteins, particularly those having thesequences set forth therein, particularly, but not limited to, thosedenoted therein: 9H7; 18B2; 2D8; 2E11; 16E1; and 22B3, including theOPGL specific antibodies having either the light chain of SEQ ID NO: 2as set forth therein in FIG. 2 and/or the heavy chain of SEQ ID NO:4, asset forth therein in FIG. 4, each of which is individually andspecifically incorporated by reference herein in its entirety fully asdisclosed in the foregoing publication. Acid and base titrations of anOPGL specific antibody (“Ab-hOPGL”) over the pH ranges of 4.5 to 5.0 and5.0 to 5.5 are described in the Examples below. The calculation ofbuffer capacity of Ab-hOPGL in these pH ranges also is described in theExamples below.

Myostatin binding agents or peptibodies, including myostatin specificpeptibodies, particularly those described in US Application PublicationNumber 2004/0181033, which is incorporated by reference herein in itsentirely particularly in parts pertinent to myostatin specificpeptibodies, including but not limited to peptibodies of the mTN8-19family, including those of SEQ ID NOS: 305-351, including TN8-19-1through TN8-19-40, TN8-19 con1 and TN8-19 con2; peptibodies of the mL2family of SEQ ID NOS: 357-383; the mL15 family of SEQ ID NOS: 384-409;the mL17 family of SEQ ID NOS: 410-438; the mL20 family of SEQ ID NOS:439-446; the mL21 family of SEQ ID NOS: 447-452; the mL24 family of SEQID NOS: 453-454; and those of SEQ ID NOS: 615-631, each of which isindividually and specifically incorporated by reference herein in itsentirety fully as disclosed in the foregoing publication.

IL-4 receptor specific antibodies, particularly those that inhibitactivities mediated by binding of IL-4 and/or IL-13 to the receptor,including those described in International Publication No. WO2005/047331 of International Application Number PCT/US2004/03742, whichis incorporated herein by reference in its entirety particularly inparts pertinent to IL-4 receptor specific antibodies, particularly suchantibodies as are described therein, particularly, and withoutlimitation, those designated therein: L1H1; L1H2; L1H3; L1H4; L1H5;L1H6; L1H7; L1H8; L1H9; L1H10; L1H11; L2H1; L2H2; L2H3; L2H4; L2H5;L2H6; L2H7; L2H8; L2H9; L2H10; L2H11; L2H12; L213; L2H14; L3H1; L4H1;L5H1; L6H1, each of which is individually and specifically incorporatedby reference herein in its entirety fully as disclosed in the foregoingpublication. Acid and base titrations over the pH ranges of 4.5 to 5.0and 5.0 to 5.5, and the calculation of buffer capacity in this range ofan IL-4 receptor specific antibody (“Ab-hIL4R”) are described in theExamples below.

Interleukin 1-receptor 1 (“IL1-R1”) specific antibodies, peptibodies andrelated proteins and the like, including but not limited to thosedescribed in U.S. Application Publication Number US2004/097712A1 whichis incorporated herein by reference in its entirety in parts pertinentto IL1-R1 specific binding proteins, monoclonal antibodies inparticular, especially, without limitation, those designated therein:15CA, 26F5, 27F2, 24E12, and 10H7, each of which is individually andspecifically incorporated by reference herein in its entirety fully asdisclosed in the aforementioned U.S. application publication.

Ang2 specific antibodies and peptibodies and related proteins and thelike, including but not limited to those described in InternationalPublication Number WO 03/057134 and U.S. Application Publication NumberUS2003/0229023, each of which is incorporated herein by reference in itsentirety particularly in parts pertinent to Ang2 specific antibodies andpeptibodies and the like, especially those of sequences describedtherein and including but not limited to: L1(N); L1(N) WT; L1(N) 1K WT;2xL1(N); 2xL1(N) WT; Con4 (N), Con4 (N) 1K WT, 2xCon4 (N) 1K; L1(C);L1(C) 1K; 2xL1 (C); Con4 (C); Con4 (C) 1K; 2xCon4 (C) 1K; Con4-L1 (N);Con4-L1 (C); TN-12-9 (N); C17 (N); TN8-8(N); TN8-14 (N); Con 1 (N), alsoincluding anti-Ang 2 antibodies and formulations such as those describedin International Publication Number WO 2003/030833 which is incorporatedherein by reference in its entirety as to the same, particularly Ab526;Ab528; Ab531; Ab533; Ab535; Ab536; Ab537; Ab540; Ab543; Ab544; Ab545;Ab546; A551; Ab553; Ab555; Ab558; Ab559; Ab565; AbF1AbFD; AbFE; AbFJ;AbFK; AbG1D4; AbGC1E8; AbH1C12; Ab1A1; Ab1F; Ab1KAb1P; and Ab1P, intheir various permutations as described therein, each of which isindividually and specifically incorporated by reference herein in itsentirety fully as disclosed in the foregoing publication.

NGF specific antibodies, including, in particular, but not limited tothose described in US Application Publication Number US2005/0074821,which is incorporated herein by reference in its entirety particularlyas to NGF-specific antibodies and related proteins in this regard,including in particular, but not limited to, the NGF-specific antibodiestherein designated 4D4, 4G6, 6H9, 7H2, 14D10 and 14D11, each of which isindividually and specifically incorporated by reference herein in itsentirety fully as disclosed in the foregoing publication,

CD22 specific antibodies and related proteins, such as those describedin U.S. Pat. No. 5,789,554 which is incorporated herein by reference inits entirety as to CD22 specific antibodies and related proteins,particularly human CD22 specific antibodies, such as but not limited tohumanized and fully human antibodies, including but not limited tohumanized and fully human monoclonal antibodies, particularly includingbut not limited to human CD22 specific IgG antibodies, such as, forinstance, a dimer of a human-mouse monoclonal hLL2 gamma-chain disulfidelinked to a human-mouse monoclonal hLL2 kappa-chain, including, butlimited to, for example, the human CD22 specific fully humanizedantibody in Epratuzumab, CAS registry number 501423-23-0. Illustrativeof the invention, acid and base titrations of a CD22-specific antibody(“Ab-hCD22”) over the pH ranges of 4.5 to 5.0 and 5.0 to 5.5 aredescribed in the Examples below. The calculation of buffer capacity ofAb-hCD22 in these pH ranges also is described in the Examples below.

IGF-1 receptor specific antibodies and related proteins such as thosedescribed in International Patent Application Number PCT/US2005/046493,which is incorporated herein by reference in its entirety as to IGF-1receptor specific antibodies and related proteins, including but notlimited to the IGF-1 specific antibodies therein designated L1H1, L2H2,L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12,L13H13, L14h14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21,L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30,L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39,L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48,L49H49, L50H50, L51H51, and L52H52, each of which is individually andspecifically incorporated by reference herein in its entirety fully asdisclosed in the foregoing International Application.

B-7 related protein 1 (“B7RP-1”) specific antibodies, (B7RP-1 also isreferred to in the literature as B7H2, ICOSL, B7h, and CD275)particularly B7RP-specific fully human monoclonal IgG2 antibodies,particularly fully human IgG2 monoclonal antibody that binds an epitopein the first immunoglobulin-like domain of B7RP-1, especially those thatinhibit the interaction of B7RP-1 with its natural receptor, ICOS, onactivated T cells in particular, especially, in all of the foregoingregards, those disclosed in U.S. Provisional Application No. 60/700,265,filed 18 Jul. 2005, which is incorporated herein by reference in itsentirety as to such antibodies and related proteins, including but notlimited to antibodies designated therein as follow: 16H (having lightchain variable and heavy chain variable sequences SEQ ID NO:1 and SEQ IDNO:7 respectively therein); 5D (having light chain variable and heavychain variable sequences SEQ ID NO:2 and SEQ ID NO:9 respectivelytherein); 2H (having light chain variable and heavy chain variablesequences SEQ ID NO:3 and SEQ ID NO:10 respectively therein); 43H(having light chain variable and heavy chain variable sequences SEQ IDNO:6 and SEQ ID NO:14 respectively therein); 41H (having light chainvariable and heavy chain variable sequences SEQ ID NO:5 and SEQ ID NO:13respectively therein); and 15H (having light chain variable and heavychain variable sequences SEQ ID NO:4 and SEQ ID NO:12 respectivelytherein), each of which is individually and specifically incorporated byreference herein, in its entirety fully as disclosed in the foregoingU.S. Provisional Application. Acid and base titrations and determinationof buffer capacity of a B7RP-1 specific antibody (“Ab-hB7RP1”) areillustrated in the Examples below.

IL-15 specific antibodies, peptibodies and related proteins, such as, inparticular, humanized monoclonal antibodies, particularly antibodiessuch as those disclosed in U.S. Application Publication Numbers:US2003/0138421; US20031023586; US2004/0071702, each of which isincorporated herein by reference in its entirety as to IL-15 specificantibodies and related proteins, including peptibodies, includingparticularly, for instance, but not limited to, HuMax IL-15 antibodiesand related proteins, such as, for instance, 146B7.

IFN gamma specific antibodies, especially human IFN gamma specificantibodies, particularly fully human anti-IFN gamma antibodies, such as,for instance, those described in US Application Publication NumberUS2005/0004353, which is incorporated herein by reference in itsentirety as to IFN gamma specific antibodies, particularly, for example,the antibodies therein designated 1118; 1118*; 1119; 1121; and 1121*each of which is individually and specifically incorporated by referenceherein in its entirety fully as disclosed in the foregoing USApplication Publication,

TALL-1 specific antibodies and other TALL specific binding proteins suchas those described in U.S. Application Publication Number 2003/0195156which is incorporated herein by reference in its entirety as to TALL-1binding proteins, particularly the molecules of Tables 4 and 5B, each ofwhich is individually and specifically incorporated by reference hereinin its entirety fully as disclosed in the foregoing US ApplicationPublication.

Stem Cell Factor(s) (“SCF”) and related proteins such as those describedin U.S. Pat. Nos. 6,204,363 and 6,207,802, each of which is incorporatedherein by reference in its entirety as to stern cell factors and relatedproteins, particularly, for example, the stem cells factor “STEMGEN™.”

Flt3-Ligands, (“Flt3L”) and related proteins such as those described inU.S. Pat. No. 6,632,424 which is incorporated herein by reference as toFlt3-ligands and related proteins in this regard.

IL-17 receptors and related proteins (“IL-17R”), such as those describedin U.S. Pat. No. 6,072,033 which is incorporated herein by reference asto Flt3-ligands and related proteins in this regard.

Etanercept, also referred to as Embrel, and related proteins.

Actimmune (Interferon-gamma-1b), Activase (Alteplase), Aldurazme(Laronidase), Amevive (Alefacept), Avonex (Interferon beta-1a), BeneFIX(Nonacog alfa), Beromun (Tasonermin), Beatseron (Interferon-beta-1b),BEXXAR (Tositumomab), Tev-Tropin (Somatropin), Bioclate or RECOMBINATE(Recombinant), CEREZME (Imiglucerase), ENBREL (Etanercept), Eprex(epoetin alpha), EPOGEN/Procit (Epoetin alfa), FABRAZYME (Agalsidasebeta), Fasturtec/Elitek ELITEK (Rasburicase), FORTEO (Teriparatide),GENOTROPIN (Somatropin), GlucaGen (Glucagon), Glucagon (Glucagon, rDNAorigin), GONAL-F (follitropin alfa), KOGENATE FS (Octocog alfa),HERCEPTIN (Trastuzumab), HUMATROPE (SOMATROPIN), HUMIRA (Adalimumab),Insulin in Solution, INFERGEN® (Interferon alfacon-1), KINERET®(anakinra), Kogenate FS (Antihemophilic Factor), LEUKIN (SARGRAMOSTIMRecombinant human granulocyte-macrophage colony stimulating factor(rhuGM-CSF)), CAMPATH (Alemtuzumab), RITUXAN® (Rituximab), TNKase(Tenecteplase), MYLOTARG (gemtuzumab ozogamicin), NATRECOR (nesiritide),ARANESP (darbepoetin alfa) NEULASTA (pegfilgrastim), NEUMEGA(oprelvekin), NEUPOGEN (Filgrastim), NORDITROPIN CARTRIDGES(Somatropin), NOVOSEVEN (Eptacog alfa), NUTROPIN AQ (somatropin),Oncaspar (pegaspargase), ONTAK (denileukin diftitox), ORTHOCLONE OKT(muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS(peginterferon alfa-2a), PROLEUKIN (Aldesleukin), PULMOZYME (dornasealfa), Retavase (Reteplase), REBETRON Combination Therapy containingREBETOL® (Ribavirin) and INTRON® A (Interferon alfa-2b), REBIF(interferon beta-1a), REFACTO (Antihemophilic Factor), REFLUDAN(lepirudin), REMICADE (infliximab), REOPRO (abciximab)ROFERON®-A(Interferon alfa-2a), SIMULECT (baasiliximab), SOMAVERT (Pegivisomant),SYNAGIS® (palivizumab), Stemben (Ancestim, Stem cell factor), THYROGEN,INTRON® A (Interferon alfa-2b), PEG-INTRON® (Peginterferon alfa-2b),XIGRIS® (Drotrecogin alfa activated), XOLAIR® (Omalizumab), ZENAPAX®(daclizumab), ZEVALIN® (Ibritumomab Tiuxetan).

d. Sequence Variation

Particularly preferred proteins in regard to all of the foregoing andthe following, include those that comprise a region that is 70% or more,especially 80% or more, more especially 90% or more, yet more especially95% or more, particularly 97% or more, more particularly 98% or more,yet more particularly 99% or more identical in amino acid sequence to areference amino acid sequence of a binding protein, as illustratedabove, particularly a pharmaceutical binding protein, such as a GenBankor other reference sequence of a reference protein.

Identity in this regard can be determined using a variety of well-knownand readily available amino acid sequence analysis software. Preferredsoftware includes those that implement the Smith-Waterman algorithms,considered a satisfactory solution to the problem of searching andaligning sequences. Other algorithms also may be employed, particularlywhere speed is an important consideration. Commonly employed programsfor alignment and homology matching of DNAs, RNAs, and polypeptides thatcan be used in this regard include FASTA, TFASTA, BLASTN, BLASTP,BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being animplementation of the Smith-Waterman algorithm for execution onmassively parallel processors made by MasPar.

The BLASTN, BLASTX, and BLASTP programs are among preferred programs forsuch determinations, the former for polynucleotide sequence comparisonsand the latter two for polypeptide sequence comparisons: BLASTX forcomparison of the polypeptide sequences from all three reading frames ofpolynucleotide sequence and BLASTP for a single polypeptide sequence.

BLAST provides a variety of user definable parameters that are setbefore implementing a comparison. Some of them are more readily apparentthan others on graphical user interfaces, such as those provided by NCBIBLAST and other sequence alignment programs that can be accessed on theinternet. The settings and their values are set out and explained on theservice web sites and are explained and set out in particular detail ina variety of readily available texts, including but not limited toBIOINFORMATICS: SEQUENCE AND GENOME ANALYSIS, 2^(nd) Ed., David W.Mount, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2004), especially Chapters 3, 4, 5, and 6 as to comparison of proteinand nucleic acid sequences in general and as to BLAST comparisons andsearches in particular; SEQUENCE ANALYSIS IN A NUTSHELL: A GUIDE TOCOMMON TOOLS AND DATABASES, Scott Markel and Darryl León, O'Reilly &Associates, Sebastopol, Calif. (2003), especially Chapter 7 as to BLASTin particular, each of which is herein incorporated by reference in itsentirety particularly in parts pertinent to comparison of nucleotide andpolypeptide sequences and to determining their degree of identity,similarity, homology and/or the like, especially as to comparison of atest sequence and a reference sequence to calculate a degree (percent)of identity between them.

In preferred embodiments of the invention in this regard, relatedness ofsequences is defined as the identity score in percent returned by anyone or another of the aforementioned BLAST comparison searches with e=10and all other parameters set to their default values on the NCBI webserver as set forth in SEQUENCE ANALYSIS IN A NUTSHELL: A GUIDE TOCOMMON TOOLS AND DATABASES, Scott Markel and Darryl León, O'Reilly &Associates, Sebastopol, Calif. (2003), pages 47-51 which areincorporated herein by reference in their entireties and in allparticulars of the preferred settings for parameters of the presentinvention for comparing sequences using BLAST, such as those on NCBIBLAST.

The following references provide additional information on sequencecomparisons in this regard, and in others. GUIDE TO HUMAN GENOMECOMPUTING, Ed. Martin J. Bishop, Academic Press, Harcourt Brace &Company Publishers, New York (1994), which is incorporated herein byreference in its entirety with regard to the foregoing, particularly inparts pertinent to determining identity and or homology of amino acid orpolynucleotide sequences, especially Chapter 7. The BLAST programs aredescribed in Altschul et al., “Basic Local Alignment Research Tool,” JMol Biol 215: 403-410 (1990), which is incorporated by reference hereinin its entirety. Additional information concerning sequence analysis andhomology and identity determinations are provided in, among many otherreferences well-known and readily available to those skilled in the art:NUCLEIC ACID AND PROTEIN SEQUENCE ANALYSIS: A PRACTICAL APPROACH, Eds.M. J. Bishop and C. J. Rawings, IRL Press, Oxford, UK (1987); PROTEINSTRUCTURE: A PRACTICAL APPROACH, Ed. T. E. Creighton, IRL Press, Oxford,UK (1989); Doolittle, R. F.: “Searching through sequence databases,” MetEnz. 183: 99-110 (1990); Meyers and Miller: “Optimal alignments inlinear space” Comput. Applica. in Biosci 4: 11-17 (1988); Needleman andWunsch: “A general method applicable to the search for similarities inamino acid sequence of two proteins,” J Mol Biol 48: 443-453 (1970) andSmith and Waterman “Identification of common molecular subsequences,” JMol Biol 147: 1950 et seq. (1981), each of which is incorporated hereinby reference in its entirety with reference to the foregoing,particularly in parts pertinent to sequence comparison and identity andhomology determinations.

Particularly preferred embodiments in this regard have 50% to 150% ofthe activity of the aforementioned reference protein, particularlyhighly preferred embodiments in this regard have 60% to 125% of theactivity of the reference protein, yet more highly preferred embodimentshave 75% to 110% of the activity of the reference protein, still morehighly preferred embodiments have 85% to 125% the activity of thereference, still more highly preferred embodiments have 90% to 110% ofthe activity of the reference.

4. Formulations

Many reagents and methods conventionally employed for the formulation ofprotein pharmaceuticals can be used for the formulation ofself-buffering protein compositions in accordance with various aspectsand preferred embodiments of the invention. However, in self-bufferingprotein formulations in accordance with the invention, buffering isprovided substantially entirely by the protein itself, not by abuffering agent, as is the case with conventional formulations.Moreover, self-buffering protein formulations in accordance with variousaspects and preferred embodiments of the invention are substantiallyfree of such buffering agents.

In many other respects, however, self-buffering protein compositions inaccordance with various aspects and embodiments of the invention can beformulated using reagents and methods conventionally employed for theformulation of proteins, in particular, reagents and methods employedfor the formulation of pharmaceuticals, including pharmaceuticals forveterinary and human use, especially those reagents and methods suitablefor formulating protein pharmaceuticals for veterinary and especiallyfor human use.

In accordance therewith, many methods and ingredients for formulatingand using pharmaceuticals that are well-known and routine in thepertinent arts can be used in designing, making, and usingself-buffering protein formulations in accordance with various aspectsand preferred embodiments of the invention relating thereto. Suchmethods and ingredients are described in, to name just a few readilyavailable references in this regard, REMINGTON: THE SCIENCE AND PRACTICEOF PHARMACY, 21^(st) Ed.; Beringer et al. Editors, Lippincott, Williams& Wilkins, Philadelphia, Pa. (2005); ANSEL'S PHARMACEUTICAL DOSAGE FORMSAND DRUG DELIVERY SYSTEMS, 8^(th) Ed., Allen et al., Editors,Lippincott, Williams & Wilkins, Philadelphia, Pa. (2005); andPHARMACEUTICAL FORMULATION OF PEPTIDES AND PROTEINS, Sven Frokjaer andLars Hovgaard, Editors, CRC Press, Boca Raton, Fla. (2000), each ofwhich is herein incorporated in its entirety particularly in partspertinent to conventional ingredients and methods that may be used inself-buffering formulations of proteins in accordance with variousaspects and preferred embodiments of the invention relating thereto.

Additional methods and ingredients that can be useful in this regard aredisclosed in, among others, U.S. Pat. No. 6,171,586; WO 2005/044854;U.S. Pat. No. 6,288,030; U.S. Pat. No. 6,267,958; WO 2004/055164; U.S.Pat. No. 4,597,966; US 2003/0138417; U.S. Pat. No. 6,252,055; U.S. Pat.No. 5,608,038; U.S. Pat. No. 6,875,432; US 2004/0197324; WO 02/096457;U.S. Pat. No. 5,945,098; U.S. Pat. No. 5,237,054; U.S. Pat. No.6,485,932; U.S. Pat. No. 6,821,515; U.S. Pat. No. 5,792,838; U.S. Pat.No. 5,654,403; U.S. Pat. No. 5,908,826; EP 0 804 163; and WO2005/063291, each of which is incorporated herein by reference in itsentirety particularly in parts pertinent to pharmaceutically acceptableself-buffering protein formulations in accordance with the invention.

Various specific aspects of the ingredients and specific types offormulations are further described below, by way of illustration. Thedescription thus provided is not exhaustive of the methods andcompositions possible for self-buffering protein formulations inaccordance with the various aspects and embodiments of the invention,nor is it in any way exclusive.

In preferred embodiments of a variety of aspects of the invention,formulations of self-buffering proteins comprise a protein and acarrier, which also may be referred to herein variously, as the case maybe, as one or more of: a vehicle, a primary vehicle, a diluent, aprimary diluent, a primary carrier, a solvent and/or a primary solvent.In the broadest sense the carrier may be a gas, a liquid, or a solid, assuits the phase of the composition and/or its use(s). In someembodiments of the invention in this regard, the carrier is a solid,such as a powder in which a protein may be dispersed. In preferredembodiments in this regard, the carrier is a liquid, particularly aliquid in which the self-buffering protein is highly soluble,particularly at concentrations that provide the desired buffer capacity.Liquid carriers may be organic or non-organic. Preferably they areaqueous, most preferably they are largely or entirely comprised of purewater.

It will be appreciated that formulations for pharmaceutical use inaccordance with various aspects and embodiments of the invention must becompatible with the processes and conditions to which they will besubjected, such as, for instance, sterilization procedures (generallyapplied before mixing with an active agent), and conditions duringstorage.

Almost invariably, formulations in accordance with numerous aspects andembodiments of the invention will contain additional ingredientsincluding but not limited in any way to excipients and otherpharmaceutical agents. Nevertheless, it is to be understood thatformulations in accordance with the invention are self-bufferingformulations in which the buffer capacity is provided substantially orentirely by the primary protein itself, as described elsewhere herein.

Formulations in accordance with various aspects and embodiments of theinvention may contain, among others, excipients, as described below,including but not limited to ingredients for modifying, maintaining, orpreserving, for example, osmolality, osmolarity, viscosity, clarity,color, tonicity, odor, sterility, stability, rate of dissolution orrelease, adsorption or penetration of the formulations and/or primarypolypeptide and/or protein.

Formulations will, of course, depend upon, for example, the particularprotein being formulated, the other active agents, such as otherpharmaceuticals, that will be comprised in the formulation, the intendedroute of administration, the method of administration to be employed,the dosage, the dosing frequency, and the delivery format, among others.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide compositions comprising aprotein preferably a pharmaceutical protein and a solvent, the proteinhaving a buffer capacity per unit volume of at least that ofapproximately: 2.0 or 3.0 or 4.0 or 5.0 or 6.50 or 8.00 or 10.0 or 15.0or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or250 or 300 or 350 or 400 or 500 or 700 or 1,000 or 1,500 or 2,000 or2,500 or 3,000 or 4,000 or 5,000 mM sodium acetate buffer as determinedover the range of pH 5.0 to 4.0 pH or 5.0 to 5.5 as described in Example1 or 2 and elsewhere herein.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions, wherein,exclusive of the buffer capacity of the protein, the buffer capacity perunit volume of the composition is equal to or less than that of 1.0 or1.5 or 2.0 or 3.0 or 4.0 or 5.0 mM sodium acetate buffer as determinedover the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 as described, inExample 1 or 2 and elsewhere herein.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, wherein at the pH of the compositionthe buffer capacity of the protein is at least approximately: 1.00 or1.50 or 1.63 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or15.0 or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200or 250 or 300 or 350 or 400 or 500 or 700 or 1,000 or 1,500 or 2,000 or2,500 or 3,000 or 4,000 or 5,000 mEq per liter and per change in pH ofone pH unit.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, wherein at the pH of thecomposition, exclusive of the protein, the buffer capacity per unitvolume of the composition is equal to or less than that of a 0.50 or1.00 or 1.50 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or20.0 or 25.0 mM acetate buffer as determined over the range of pH 5.0 to4.0 or pH 5.0 to 5.5 as described in Example 1 or 2 and elsewhereherein.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, wherein at a desired pH, the proteinprovides at least approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 99.5% of the buffer capacity of the composition.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, A, wherein the concentration of theprotein is between approximately: 20 and 400, or 20 and 300, or 20 and250, or 20 and 200, or 20 and 150 mg/ml.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, wherein the pH maintained by thebuffering action of the protein is a pH between approximately: 3.5 and8.0, or 4.0 and 6.0, or 4.0 and 5.5, or 4.5 and 5.5.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, wherein the salt concentration isless than: 150 mM or 125 mM or 100 mM or 75 mM or 50 mM or 25 mM.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, and further comprising one or morepharmaceutically acceptable salts; osmotic balancing agents (tonicityagents); anti-oxidants; antibiotics; antimycotics; hulking agents;lyoprotectants; anti-foaming agents; chelating agents; preservatives;colorants; analgesics; or additional pharmaceutical agents.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, and further comprising one or morepharmaceutically acceptable polyols in an amount that is hypotonic,isotonic, or hypertonic, preferably approximately isotonic, particularlypreferably isotonic, especially preferably any one or more of sorbitol,mannitol, sucrose, trehalose, or glycerol, particularly especiallypreferably approximately 5% sorbitol, 5% mannitol, 9% sucrose, 9%trehalose, or 2.5% glycerol, very especially in this regard 5% sorbitol,5% mannitol, 9% sucrose, 9% trehalose, or 2.5% glycerol.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, and further comprising one or morepharmaceutically acceptable surfactants, preferably one or more ofpolysorbate 20, polysorbate 80, other fatty acid esters of sorbitan,polyethoxylates, and poloxamer 188, particularly preferably polysorbate20 or polysorbate 80, preferably approximately 0.001 to 0.1% polysotbate20 or polysorbate 80, very preferably approximately 0.002 to 0.02%polysorbate 20 or polysorbate 80, especially 0.002 to 0.02% polysorbate20 or polysorbate 80.

Formulations in accordance with certain of the preferred embodiments invarious aspects of the invention provide self-buffering proteincompositions, particularly pharmaceutical protein compositions,comprising a protein and a solvent, wherein the protein is apharmaceutical agent and the composition is a sterile formulationthereof suitable for treatment of a veterinary or a human medicalsubject.

Also among formulations in accordance with various aspects andembodiments of the invention herein described are lyophilizedcompositions in accordance with the foregoing, particularly lyophilizedcompositions that when reconstituted provide a formulation as describedabove and elsewhere herein.

a. Excipients and Other Additional Ingredients

As discussed above, certain embodiments in accordance with aspects ofthe invention provide self-buffering protein compositions, particularlypharmaceutical protein compositions, that comprise, in addition to theprotein, particularly a pharmaceutical protein, one or more excipientssuch as those illustratively described in this section and elsewhereherein. Excipients can be used in the invention in this regard for awide variety of purposes, such as adjusting physical, chemical, orbiological properties of formulations, such as adjustment of viscosity,and or processes of the invention to improve effectiveness and or tostabilize such formulations and processes against degradation andspoilage due to, for instance, stresses that occur during manufacturing,shipping, storage, pre-use preparation, administration, and thereafter.

A variety of expositions are available on protein stabilization andformulation materials and methods useful in this regard, such as Arakawaet al., “Solvent interactions in pharmaceutical formulations,” PharmRes. 8(3): 285-91 (1991); Kendrick et al., “Physical stabilization ofproteins in aqueous solution,” in: RATIONAL DESIGN OF STABLE PROTEINFORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds.Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al.,“Surfactant-protein interactions,” Pharm Biotechnol. 13: 159-75 (2002),each of which is herein incorporated by reference in its entirety,particularly in parts pertinent to excipients and processes of the samefor self-buffering protein formulations in accordance with the currentinvention, especially as to protein pharmaceutical products andprocesses for veterinary and/or human medical uses.

Various excipients useful in the invention are listed in Table 1 andfurther described below.

TABLE 1 Types of Excipients and Their Functions Function Type LiquidsLyophilates Tonicity Provides isotonicity to the formulation Stabilizersinclude cryo and Agents/ such that it is suitable for injectionlyoprotectants Stabilizers Examples include polyols, salts, and Examplesinclude polyols, sugars and amino acids polymers Help maintain theprotein in a more Cryoprotectants protect proteins from compact state(polyols) freezing stresses Minimize electrostatic, solution protein-Lyoprotectants stabilize proteins in the protein interactions (salts)freeze-dried state Bulking Not applicable Used to enhance productelegance and to Agents prevent blowout Provides structural strength tothe lyo cake Examples include mannitol and glycine SurfactantsPrevent/control aggregation, particle Employed if aggregation during theformation and surface adsorption of drug lyophilization process is anissue Examples include polysorbate 20 and 80 May serve to reducereconstitution times Examples include polysorbate 20 and 80Anti-oxidants Control protein oxidation Usually not employed, molecularreactions in the lyophilized cake are greatly retarded Metal A specificmetal ion is included in a May be included if a specific metal ion isIons/ liquid formulation only as a co-factor included only as aco-factor Chelating Divalent cations such as zinc and Chelating agentsare generally not Agents magnesium are utilized in suspension needed inlyophilized formulations formulations Chelating agents are used toinhibit heavy metal ion catalyzed reactions Preservatives Importantparticularly for multi-dose For multi-dose formulations onlyformulations Provides protection against microbial Protects againstmicrobial growth, growth in formulation Example: benzyl alcohol Isusually included in the reconstitution diluent (e.g. bWFI)

i. Salts

Salts may be used in accordance with certain of the preferredembodiments of the invention to, for example, adjust the ionic strengthand/or the isotonicity of a self-buffering formulation and/or to improvethe solubility and/or physical stability of a self-buffering protein orother ingredient of a self-buffering protein composition in accordancewith the invention.

As is well known, ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface and by shieldingcharged and polar groups in the protein and reducing the strength oftheir electrostatic interactions, attractive, and repulsiveinteractions. Ions also can stabilize the denatured state of a proteinby binding to, in particular, the denatured peptide linkages (—CONH) ofthe protein. Furthermore, ionic interaction with charged and polargroups in a protein also can reduce intermolecular electrostaticinteractions and, thereby, prevent or reduce protein aggregation andinsolubility.

Ionic species differ significantly in their effects on proteins. Anumber of categorical rankings of ions and their effects on proteinshave been developed that can be used in formulating self-bufferingprotein compositions in accordance with the invention. One example isthe Hofmeister series, which ranks ionic and polar non-ionic solutes bytheir effect on the conformational stability of proteins in solution.Stabilizing solutes are referred to as “kosmotropic,” Destabilizingsolutes are referred to as chaotropic. Kosmotropes commonly are used athigh concentrations (e.g., >1 molar ammonium sulfate) to precipitateproteins from solution (“salting-out”). Chaotropes commonly are used todenture and/or to solubilize proteins (“salting-in”). The relativeeffectiveness of ions to “salt-in” and “salt-out” defines their positionin the Hofmeister series.

In addition to their utilities and their drawbacks (as discussed above)salts also are effective for reducing the viscosity of proteinformulations and can be used in the invention for that purpose.

In order to maintain isotonicity in a parenteral formulation inaccordance with preferred embodiments of the invention, improve proteinsolubility and/or stability, improve viscosity characteristics, avoiddeleterious salt effects on protein stability and aggregation, andprevent salt-mediated protein degradation, the salt concentration inself-buffering formulations in accordance with various preferredembodiments of the invention are less than 150 mM (as to monovalentions) and 150 mEq/liter for multivalent ions. In this regard, in certainparticularly preferred embodiments of the invention, the total saltconcentration is from about 75 mEq/L, to about 140 mEq/L.

ii. Amino Acids

Free amino acids can be used in protein formulations in accordance withvarious preferred embodiments of the invention as, to name a few,bulking agents, stabilizers and antioxidants. However, amino acidscomprised in self-buffering protein formulations in accordance with theinvention do not provide buffering action. For this reason, those withsignificant buffer capacity either are not employed, are not employed atany pH around which they have significant buffering activity, or areused at low concentration so that, as a result, their buffer capacity inthe formulation is not significant. This is particularly the case forhistidine and other amino acids that commonly are used as buffers inpharmaceutical formulations.

Subject to the foregoing consideration, lysine, proline, serine, andalanine can be used for stabilizing proteins in a formulation. Glycineis useful in lyophilization to ensure correct cake structure andproperties. As a result it is a common ingredient in lyophilizedformulations and reconstituted lyophilates, such as Neumega®,Genotropin®, and Humatrope®. Arginine may be useful to inhibit proteinaggregation, in both liquid and lyophilized formulations, such asActivase®, Avonex®, and Enbrel® liquid. Methionine is useful as anantioxidant.

iii. Polyols

Polyols include sugars, e.g., mannitol, sucrose, and sorbitol andpolyhydric alcohols such as, for instance, glycerol and propyleneglycol, and, for purposes of discussion herein, polyethylene glycol(PEG) and related substances. Polyols are kosmotropic. They are usefulstabilizing agents in both liquid and lyophilized formulations toprotect proteins from physical and chemical degradation processes.Polyols also are useful for adjusting the tonicity of formulations.

Among polyols useful in the invention in this regard, is mannitol,commonly used to ensure structural stability of the cake in lyophilizedformulations, such as, for example Leukine®, Enbrel®—Lyo, andBetaseron®. It ensures structural stability to the cake. It is generallyused with a lyoprotectant, e.g., sucrose. Sorbitol and sucrose are amongpreferred agents for adjusting tonicity and as stabilizers to protectagainst freeze-thaw stresses during transport or the preparation ofbulks during the manufacturing process. Reducing sugars (which containfree aldehyde or ketone groups), such as glucose and lactose, canglycate surface lysine and arginine residues. Therefore, they generallyare not among preferred polyols for use in accordance with theinvention. In addition, sugars that form such reactive species, such assucrose, which is hydrolyzed to fructose and glucose under acidicconditions, and consequently engenders glycation, also is not amongpreferred amino acids of the invention in this regard. PEG is useful tostabilize proteins and as a cryoprotectant and can be used in theinvention in this regard, such as it is in Recombinate®.

iv. Surfactants

Protein molecules are susceptible to adsorption on surfaces and todenaturation and consequent aggregation at air-liquid, solid-liquid, andliquid-liquid interfaces. These effects generally scale inversely withprotein concentration. These deleterious interactions generally scaleinversely with protein concentration and typically are exacerbated byphysical agitation, such as that generated during the shipping andhandling of a product.

Surfactants routinely are used to prevent, minimize, or reduce surfaceadsorption. Useful surfactants in the invention in this regard includepolysorbate 20, polysorbate 80, other fatty acid esters of sorbitanpolyethoxylates, and poloxamer 188.

Surfactants also are commonly used to control protein conformationalstability. The use of surfactants in this regard is protein-specificsince, any given surfactant typically will stabilize some proteins anddestabilize others.

Polysorbates are susceptible to oxidative degradation and often, assupplied, contain sufficient quantities of peroxides to cause oxidationof protein residue side-chains, especially methionine. Consequently,polysorbates should be used carefully, and when used, should be employedat their lowest effective concentration. In this regard, polysorbatesexemplify the general rule that excipients should be used in theirlowest effective concentrations.

v. Antioxidants

A variety of processes can result in harmful oxidation of proteins inpharmaceutical formulations. To some extent deleterious oxidation ofproteins can be prevented in pharmaceutical formulations by maintainingproper levels of ambient oxygen and temperature and by avoiding exposureto light. Antioxidant excipients can be used as well to preventoxidative degradation of proteins. Among useful antioxidants in thisregard are reducing agents, oxygen/free-radical scavengers, andchelating agents. Antioxidants for use in therapeutic proteinformulations in accordance with the invention preferably arewater-soluble and maintain their activity throughout the shelf life of aproduct. EDTA is a preferred antioxidant in accordance with theinvention in this regard and can be used in the invention in much thesame way it has been used in formulations of acidic fibroblast growthfactor and in products such as Kineret® and Ontak®.

Antioxidants can damage proteins. For instance, reducing agents, such asglutathione in particular, can disrupt intramolecular disulfidelinkages. Thus, antioxidants for use in the invention are selected to,among other things, eliminate or sufficiently reduce the possibility ofthemselves damaging proteins in the formulation.

vi. Metal Ions

Formulations in accordance with the invention may include metal ionsthat are protein co-factors and that are necessary to form proteincoordination complexes, such as zinc necessary to form certain insulinsuspensions. Metal ions also can inhibit some processes that degradeproteins. However, metal ions also catalyze physical and chemicalprocesses that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit isomerization ofaspartic acid to isoaspartic acid. Ca⁺² ions (up to 100 mM) can increasethe stability of human deoxyribonuclease (rhDNase, Pulmozyme®). Mg⁺²,Mn⁺², and Zn⁺², however, can destabilize rhDNase. Similarly, Ca⁺² andSr⁺² can stabilize Factor VIII, it can be destabilized by Mg⁺², Mn⁺² andZn⁺², Cu⁺² and Fe^(+2,) and its aggregation can be increased by Al⁺³ions.

vii. Preservatives

Preservatives are necessary when developing multi-dose parenteralformulations that involve more than one extraction from the samecontainer. Their primary function is to inhibit microbial growth andensure product sterility throughout the shelf-life or term of use of thedrug product. Commonly used preservatives include benzyl alcohol, phenoland m-cresol. Although preservatives have a long history of use withsmall-molecule parenterals, the development of protein formulations thatincludes preservatives can be challenging. Preservatives almost alwayshave a destabilizing effect (aggregation) on proteins, and this hasbecome a major factor in limiting their use in multi-dose proteinformulations. To date, most protein drugs have been formulated forsingle-use only. However, when multi-dose formulations are possible,they have the added advantage of enabling patient convenience, andincreased marketability. A good example is that of human growth hormone(hGH) where the development of preserved formulations has led tocommercialization of more convenient, multi-use injection penpresentations. At least four such pen devices containing preservedformulations of hGH are currently available on the market. Norditropin®(liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech) & Genotropin(lyophilized—dual chamber cartridge, Pharmacia & Upjohn) contain phenolwhile Somatrope® (Eli Lilly) is formulated with m-cresol.

Several aspects need to be considered during the formulation anddevelopment of preserved dosage forms. The effective preservativeconcentration in the drug product must be optimized. This requirestesting a given preservative in the dosage form with concentrationranges that confer anti-microbial effectiveness without compromisingprotein stability. For example, three preservatives were successfullyscreened in the development of a liquid formulation for interleukin-1receptor (Type 1) using differential scanning calorimetry (DSC). Thepreservatives were rank ordered based on their impact on stability atconcentrations commonly used in marketed products.

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time for which a preservative is in contact with theprotein, significantly minimizing the associated stability risks. Withliquid formulations, preservative effectiveness and stability have to bemaintained over the entire product shelf-life (˜18 to 24 months). Animportant point to note is that preservative effectiveness has to bedemonstrated in the final formulation containing the active drug and allexcipient components.

Self-buffering protein formulations in accordance with the invention,particularly self-buffering biopharmaceutical protein formulations,generally will be designed for specific routes and methods ofadministration, for specific administration dosages and frequencies ofadministration, for specific treatments of specific diseases, withranges of bio-availability and persistence, among other things.

Formulations thus may be designed in accordance with the invention fordelivery by any suitable route, including but not limited to orally,aurally, opthalmically, rectally, and vaginally, and by parenteralroutes, including intravenous and intraarterial injection, intramuscularinjection, and subcutaneous injection.

b. Formulations for Parenteral Administration

Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules using one or more of the carriers or diluents mentioned for usein the formulations for oral administration or by using other suitabledispersing or wetting agents and suspending agents.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired protein in a pharmaceutically acceptable vehicle. A particularlysuitable vehicle for parenteral injection is sterile pure water in whichthe protein is formulated as a sterile, isotonic self-bufferingsolution.

Such preparations may also involve the formulation of the desiredprotein in the form of, among other things, injectable microspheres,bio-erodible particles, polymeric compounds (polylactic acid,polyglycolic acid), beads, or liposomes, including those that providefor controlled or sustained release. Such formulations may be introducedby implantable drug delivery devices, among others.

Formulations for parenteral administration also may contain substancesthat adjust the viscosity, such as carboxymethyl cellulose, sorbitol,and dextran. Formulations may also contain ingredients that increasesolubility of the desired protein or other ingredients and those thatstabilize one or more such ingredients, including in some cases, theself-buffering protein.

c. Formulations for Pulmonary Administration

A pharmaceutical composition in accordance with certain embodiments ofthe invention may be suitable for inhalation. For pulmonaryadministration, the pharmaceutical composition may be administered inthe form of an aerosol or with an inhaler including dry powder aerosol.For example, a binding agent may be formulated as a dry powder forinhalation. Inhalation solutions may also be formulated with apropellant for aerosol delivery. In yet another embodiment, solutionsmay be nebulized. Pulmonary administration is further described in PCTApplication No. PCT/US94/001875, which describes pulmonary delivery ofchemically modified proteins.

d. Formulations for Oral Administration

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, suspension, or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient. Examplesof such dosage units are tablets or capsules. Formulations for oraladministration in accordance with the invention in this regard can bemade conventionally wherein buffering in the formulation is provided bythe self-buffering protein as described elsewhere herein.

e. Controlled Release Formulations

Among additional formulations that can be useful in the invention asherein described are sustained- and controlled-delivery formulations.Techniques for making such sustained- and controlled-deliveryformulations that may be used in accordance with various aspects andpreferred embodiments of the invention are well-known to those skilledin the art. Among these are delivery methods that use liposome carriers,bio-erodible microparticles, porous beads, and semi-permeable polymermatrices, such as those described in PCT/US93/00829; U.S. Pat. No.3,773,919; EP 58,481; Sidman et al., Biopolymers, 22:547-556 (1983);Langer et al., J. Biomed. Mater. Res., 15:167-277, (1981); Langer etal., Chem. Tech., 12:98-105(1982); EP 133,988; Eppstein et al., Proc.Natl. Acad. Sci. (USA), 82:3688-3692 (1985); EP 36,676; EP 88,046; andEP 143,949, each of which is hereby incorporated by reference in itsentirety, particularly in parts pertinent to self-buffering sustained-and controlled-delivery pharmaceutical protein formulations inaccordance with the invention herein described.

f. Sterilization

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

g. Storage

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

h. Additional Pharmaceutical Agents

Self-buffering protein compositions in accordance with the invention,particularly self-buffering pharmaceutical protein compositions, cancomprise in addition to the self-buffering protein of the composition,one or more additional pharmaceutical agents. Such agents may beproteins as well, or they may be other types of agents. Included amongsuch agents are those for prevention or treatment of any disorder ordisease. Such agents include, for instance, antibiotics andantimycotics. They also include agents for treating human disorders,including but not limited to, agents for treating inflammatory diseases,cancers, metabolic disorders, neurological and renal disorders, to namejust a few. Agents that may be used in the invention in this regard alsoinclude agents useful to augment the action of a self-bufferingcomposition and or prevent, ameliorate, or treat any undesirable sideeffects of the administration thereof.

i. Methods for Making Self-Buffering Protein Formulations

Compositions in accordance with the invention may be produced usingwell-known, routine methods for making, formulating, and using proteins,particularly pharmaceutical proteins. In certain of the preferredembodiments of a number of aspects of the invention in this regard,methods for preparing the compositions comprise the use of counter ionsto remove residual buffering agents. In this regard the term counter ionis any polar or charged constituent that acts to displace buffer fromthe composition during its preparation. Counter ions useful in thisregard include, for instance, glycine, chloride, sulfate, and phosphate.The term counter ion in this regard is used to mean much the same thingas displacement ion.

Residual buffering agents can be removed using the counter ions in thisregard, using a variety of well-known methods, including but not limitedto, standard methods of dialysis and high performance membranediffusion-based methods such as tangential flow diafiltration. Methodsfor residual buffer removal employing a counter ion in this regard canalso, in some cases, be carried out using size exclusion chromatography.

In certain related preferred embodiments in this regard, compositions inaccordance with the invention are prepared by a process that involvesdialysis against a bufferless solution at a pH below that of thepreparation containing the self-buffering protein. In particularlypreferred embodiments of the invention in this regard, the bufferlesssolution comprises counter ions, particularly those that facilitateremoval of residual buffer and do not adversely affect theself-buffering protein or the formulation thereof. In furtherparticularly preferred embodiments of the invention in this regard,following dialysis the pH of the preparation is adjusted to the desiredpH using dilute acid or dilute base.

In certain related particularly preferred embodiments in this regard,compositions in accordance with the invention are prepared by a processthat involves tangential flow diafiltration against a bufferlesssolution at a pH below that of the preparation containing theself-buffering protein. In particularly preferred embodiments of theinvention in this regard, the bufferless solution comprises counterions, particularly those that facilitate removal of residual buffer anddo not adversely affect the self-buffering protein or the formulationthereof. In further particularly preferred embodiments of the inventionin this regard, following diafiltration the pH of the preparation isadjusted to the desired pH using dilute acid or dilute base.

5. Routes of Administration

Formulations in accordance with the invention, in various embodiments,may be administered by a variety of suitable routes, well-known to thoseskilled in the art of administering therapeutics to a subject. Inembodiments of the invention in this regard, one or more formulations,as described elsewhere herein, are administered via the alimentarycanal. In other embodiments one or more formulations as describedelsewhere herein are administered parenterally. In various embodimentsone or more formulations may be administered via the alimentary canal inconjunction with one or more other formulations administeredparenterally.

Such routes in a variety of embodiments include but are not limited toadministration of the compositions orally, ocularly, mucosally,topically, rectally, pulmonarily, such as by inhalation spray, andepicutaneously. The following parenteral routes of administration alsoare useful in various embodiments of the invention: administration byintravenous, intraarterial, intracardiac, intraspinal, intrathecal,intraosseous, intraarticular, intrasynovial, intracutaneous,intradermal, subcutaneous, peritoneal, and/or intramuscular injection.In some embodiments intravenous, intraarterial, intracutaneous,intradermal, subcutaneous and/or intramuscular injection are used. Insome embodiments intravenous, intraarterial, intracutaneous,subcutaneous, and/or intramuscular injection are used.

In certain embodiments of the invention the compositions areadministered locally, for instance by intraocular injection to treatocular neovascularization, retinopathy, or age-related maculardegeneration.

6. Doses

The amount of a self-buffering protein formulation administered and thedosage regimen for treating a disease condition with the formulationdepends on a variety of factors, including the age, weight, sex, andmedical condition of the subject, the type of disease, the severity ofthe disease, the route and frequency of administration, and theparticular formulation employed. In particular the amount will depend onthe protein therapeutic being administered and any other therapeuticagents being administered in conjunction therewith. Dosages can bedetermined for formulations in accordance with the invention usingwell-established routine pharmaceutical procedures for this purpose.

7. Dosing Regimens

Formulations of the invention can be administered in dosages and bytechniques well-known to those skilled in the medical and veterinaryarts taking into consideration such factors as the age, sex, weight, andcondition of the particular patient, and the formulation that will beadministered (e.g., solid vs. liquid). Doses for humans or other mammalscan be determined without undue experimentation by the skilled artisan,from this disclosure, the documents cited herein, and the knowledge inthe art.

In accordance with various embodiments, proper dosages and dosing planswill depend on numerous factors, and may vary in differentcircumstances. The parameters that will determine the optimal dosageplans to be administered typically will include some or all of thefollowing: the disease being treated and its stage; the species of thesubject, their health, gender, age, weight, and metabolic rate; othertherapies being administered; and expected potential complications fromthe subject's history or genotype.

The optimal dosing plan in a given situation also will take intoconsideration the nature of the formulation, the way it is administered,the distribution route following administration, and the rate at whichit will be cleared both from sites of action and from the subject'sbody. Finally, the determination of optimal dosing preferably willprovide an effective dose that is neither below the threshold of maximalbeneficial effect nor above the threshold where the deleterious effectsassociated with the dose of the active agents outweighs the advantagesof the increased dose.

It will be appreciated that a “dose” may be delivered all at once,fractionally, or continuously over a period of time. The entire dosealso may be delivered to a single location or spread fractionally overseveral locations. Furthermore, doses may remain the same over atreatment, or they may vary.

In various embodiments, formulations in accordance with the inventionare administered in an initial dose, and thereafter maintained byfurther administrations. A formulation of the invention in someembodiments is administered by one method initially, and thereafteradministered by the same method or by one or more different methods. Thedosages of on-going administrations may be adjusted to maintain atcertain values the levels of the active agents in the subject. In someembodiments the compositions are administered initially, and/or tomaintain their level in the subject, by intravenous injection. In avariety of embodiments, other forms of administration are used.

Formulations of the invention may be administered in many frequenciesover a wide range of times, including any suitable frequency and rangeof times that delivers a treatment-effective dose. Doses may becontinuously delivered, administered every few hours, one or more timesa day, every day, every other day or several times a week, or lessfrequently. In some embodiments they are administered over periods ofone, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, or more days. In some embodiments they areadministered over periods of one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, or more months. In a variety ofembodiments they are administered for a period of one, two, three, four,five, six, seven, eight, nine, ten, or more years. Suitable regimens forinitial administration and further doses for sequential administrationsmay all be the same or may be variable. Appropriate regimens can beascertained by the skilled artisan, from this disclosure, the documentscited herein, and the knowledge in the art. Generally lengths oftreatment will be proportional to the length of the disease process, theeffectiveness of the therapies being applied, and the condition andresponse of the subject being treated.

8 Diseases and Treatments

Self-buffering pharmaceutical protein compositions in accordance withthe invention, in preferred embodiments, are useful to treat subjectssuffering from a wide variety of disorders and diseases. As notedelsewhere herein, the invention provides, among others, self-bufferingcompositions of pharmaceutical antibodies, antibody-derivedpharmaceutical proteins, and antibody-related pharmaceutical proteins,that can comprise Fc effector functions and binding domains specific fora wide variety of disease-related targets and that are useful fortreating disease. These proteins and self-buffering compositions thereofare described at length herein above, as well as their use in treatingvarious disorders and diseases associated with their targets. Methodsfor using the compositions, including formulation methods,administration methods, doses, and dosing methods are all describedillustratively above. The formulation and administration of anyparticular composition of the invention can be tailored to the treatmentof a particular disease, using well-known and routine techniques in thearts for doing so, taken in light of the guidance provided by thepresent description of the invention. Among diseases usefully treatedusing self-buffering pharmaceutical protein formulations in accordancewith various aspects and preferred embodiments of the invention areinflammatory diseases, cancers, metabolic disorders, neurological andrenal disorders, to name just a few.

9. Packaging and Kits

The invention also provides kits comprising self-buffering proteinformulations, particularly kits comprising in one more containers, aself-buffering pharmaceutical protein formulation and instructionsregarding the use thereof, particularly such kits wherein theformulation is a pharmaceutically acceptable formulation for human use.Among preferred kits are those comprising one or more containers of aself-buffering protein formulation of the invention and one or moreseparate documents, information pertaining to the contents of the kit,and/or the use of its contents, particularly those wherein the proteinis a biopharmaceutical protein, especially those wherein the protein isa biopharmaceutical protein formulated for the treatment of a disease inhumans.

In certain aspects of the invention in this regard, preferred kitsinclude kits as above further comprising one or more single ormulti-chambered syringes (e.g., liquid syringes and lyosyringes) foradministering one or more self-buffering protein formulations of theinvention. In certain aspects of the invention in this regard, certainof the particularly preferred kits further comprise preloaded syringes.In further particularly preferred embodiments in this regard, the kitscomprise a self-buffering pharmaceutical composition for parenteraladministration, sealed in a vial under partial vacuum in a form readyfor loading into a syringe and administration to a subject. Inespecially preferred embodiments in this regard, the composition isdisposed therein under partial vacuum. In all of these regards andothers, in certain further particularly preferred embodiments the kitscontain one or more vials in accordance with any of the foregoing,wherein each vial contains a single unit dose for administration to asubject. In all these respects and others the invention, further relatesto kits comprising lyophilates, disposed as above, that uponreconstitution provide compositions in accordance therewith. In thisregard, the invention further provides in certain of its preferredembodiments, kits that contain a lyophilate in accordance with theinvention and a sterile diluent for reconstituting the lyophilate.

EXAMPLES

The present invention, is additionally described by way of the followingillustrative, non-limiting Examples.

Example 1 Acid Titrations and Buffer Capacities of Sodium AcetateBuffers in the Range pH 5.0 to 4.0

A stock solution of known concentration of acetic acid was prepared bydiluting ultrapure glacial acetic acid in HPLC grade water and thentitrating the pH up to the desired value with NaOH. Stocks wereequilibrated to the air and to 21° C. Volumetric standards were preparedat a concentration of 1 N and diluted as necessary with HPLC water.

One mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, and 15 mM sodium acetate bufferswere prepared by diluting the stock in HPLC water. The solutions weretitrated with HCl. 0.2 N HCl was used for the 1, 2.5, and 5 mMsolutions, 0.4 N HCl was used for the 7.5 mM solution, and 0.8 N HCl wasused for the 10 and 15 mM solutions. The titrations were performed usingstandard analytical laboratory techniques.

FIG. 1, Panel A shows the titration data and the least squares trendlines calculated from the data for each solution. The slope of the trendline calculated from each data set was taken as the buffer capacity ofthe corresponding acetate buffer. The linear dependence of buffercapacity on acetate buffer concentration is shown in FIG. 1, Panel B.

Example 2 Base Titrations and Buffer Capacities of Sodium AcetateBuffers in the Range pH 5.0 to 5.5

Acetate buffer stocks and solutions for titration were prepared asdescribed in Example 1. The solutions were titrated as described inExample 1, except that the solutions were titrated from pH 5.0 to 5.5and the titrations were done using NaOH instead of HCl. 0.2 N NaOH wasused to titrate the 1, 2.5, and 5 mM solutions and 0.4 N NaOH was usedfor the 7.5, 10, and 15 mM solutions. The results of the titrations areshown in FIG. 2A. The linear dependence of buffer capacity onconcentration of acetate buffer is displayed in FIG. 2B.

Example 3 Determination of Acetate by HPLC

Acetate was determined in acetate buffer samples using analyticalSE-HPLC. A standard curve for peak areas as a function of acetateconcentration was established by analysis of acetate in buffers of knownacetate concentration. The amount of acetate in test samples wasinterpolated from the standard curve. A standard curve is shown in FIG.3. Nominal and measured amount of acetate in test buffers are tabulatedbelow the standard curve in the figure.

Example 4 Acid Titrations of Ab-hOPGL Formulations Over the Range of pH5.0 to pH 4.0

Bulk Ab-hOPGL in 10 mM acetate (nominal value), 5% sorbitol, pH 5.0 wasdiafiltered against 5.25% sorbitol, pH 3.2 (adjusted with HCl) in aLABSCALE TFF® system (Millipore) with a multi-manifold cassette, using 3Millipore Pellicon XL 50 regenerated cellulose ultra-filtrationmembranes. The diafiltration solution was exchanged 8 to 10 times overthe course of the diafiltration for each formulation. Followingdiafiltration, the pH of the resulting buffer-free solution was measuredand adjusted to pH 5.0, using 0.05 N HCl or 0.05 N NaOH.

One, 10, 30, 60, 90, and 110 mg/ml solutions were prepared for titrationby dilution. The pH of each dilution was adjusted to 5.0 with NaOH orHCl as necessary. Titrations were carried out as described in theforegoing Examples. 0.2 N HCl was used to titrate the 1, 10, and 30mg/ml solutions. 0.4 N HCl was used to titrate the 60 mg/ml solution.0.8 N HCl was used to titrate the 90 and 110 solutions.

The results of the titrations are depicted in FIG. 4. The least squaresregression line is shown for the dataset for each concentration. Thebuffer capacity was taken as the slope of the regression line for eachconcentration.

Example 5 Base Titrations of Ab-hOPGL Formulations Over the Range of pH5.0 to 6.0

One, 10, 30, 60, 90, and 110 mg/ml solutions of Ab-hOPGL were preparedfor titration as described in Example 4. Base titrations were carriedout using NaOH as described in preceding Examples. 0.2 N NaOH was usedfor the 1, 10, 30, and 60 mg/ml solutions and 0.4 N NaOH was used forthe 90 and 110 mg/ml solutions. Results of the titrations are depictedin the graph in FIG. 5. Linear regression lines are shown for the datafor each concentration. The buffer capacity was taken as the slope ofthe regression line for each concentration.

Example 6 Residual Acetate Levels in Self-Buffering Ab-hOPGLFormulations

The amount of residual acetate was determined in Ab-hOPGL formulationsusing the methods described in Example 3. The results are depictedgraphically in FIG. 6, which shows a standard curve relating HPLCmeasurements to acetate concentrations and, below the graph, atabulation of the results of determinations made on Ab-hOPGLformulations at different concentrations. Ab-hOPGL concentrations areindicated on the left (“Nominal”) and the measured concentration ofacetate in each of the Ab-hOPGL concentration is indicated on the right.

Example 7 Buffer Capacity of Ab-hOPGL Formulations Plus or MinusResidual Acetate in the Range of pH 5.0 to 4.0

Self-buffered Ab-hOPGL formulations were prepared and titrated with HClas described in foregoing Examples. In addition, data was adjusted bysubtracting the contribution of residual acetate buffer based on thedetermination of acetate content by SE-HPLC as described in, forinstance, Example 3. Buffer capacities were determined as describedabove. The same analysis was carried out on both sets of data. Theresults, depicted in FIG. 7, show the effect of residual acetate on thebuffer capacity of the Ab-hOPGL preparations. The results make it clearthat the buffer capacity of residual acetate is a minor factor in thebuffer capacity of the self-buffering Ab-hOPGL formulations that wereanalyzed.

Example 8 Buffer Capacity of Ab-hOPGL Plus or Minus Residual Acetate inthe Range of pH 5.0 to 6.0

Self-buffered Ab-hOPGL formulations were prepared and titrated with NaOHas described in foregoing Examples. In addition, data was adjusted bysubtracting the contribution of residual acetate buffer based on thedetermination of acetate content by SE-HPLC as described in, forinstance, Example 3. Buffer capacities were determined as describedabove. The same analysis was carried out on both sets of data. Theresults, depicted in FIG. 8, show the effect of residual acetate on thebuffer capacity of the Ab-hOPGL preparations. The results make it clearthat the buffer capacity of residual acetate is a minor factor in thebuffer capacity of the self-buffering Ab-hOPGL formulations that wereanalyzed.

Example 9 pH and Ab-hOPGL Stability in Self-Buffered and ConventionallyBuffered Formulations

Self-buffering formulations of Ab-hOPGL were prepared as described inthe foregoing Examples. In addition, formulations were made containing aconventional buffering agent, either acetate or glutamate. Allformulations contained 60 mg/ml Ab-hOPGL. The stability of pH andAb-hOPGL in the formulations was monitored for six months of storage at4° C. Stability was monitored by determining monomeric Ab-hOPGL in theformulations over the time course of storage. The determination was madeusing SE-HPLC as described above. The results for all three formulationsare shown in FIG. 9. Panel A shows the stability of Ab-hOPGL in thethree formulations. Stability in the self-buffered formulation is asgood as in the conventionally buffered formulations. Panel B shows thepH stability of the three formulations. Again, pH stability in theself-buffered formulation is as good as in the conventionally bufferedformulations.

Example 10 Titration and Buffer Capacities of Ab-hB7RP1—pH 5.0 to 4.0

Self-buffering formulations of Ab-hB7RP1 were prepared in concentrationsof 1, 10, 30, and 60 mg/ml, as described for Ab-hOPGL in the foregoingExamples. Titrations were carried out using HCl as described above. Inaddition, data was adjusted by subtracting the contribution of residualacetate buffer based on the determination of acetate content by SE-HPLCas described in, for instance, Example 3. FIG. 10, Panel A shows thetitration results. FIG. 10, Panel B shows the dependence of buffercapacity on the concentration of Ab-hB7RP1 formulations before and aftersubtracting the contribution of residual acetate buffer. The resultsclearly show the self-buffering capacity of Ab-hB7RP1 in this pH range.At 40 mg/ml it provides approximately as much buffer capacity in this pHrange as 10 mM sodium acetate buffer. At 60 mg/ml it providesapproximately as much buffer capacity as 15 mM sodium acetate buffer.

Example 11 Titration and Buffer Capacities for Ab-hB7RP1—pH 5.0 to 6.0

Self-buffering formulations of Ab-hB7RP1 were prepared in concentrationsof 1, 10, 30, and 60 mg/ml, as described for Ab-hOPGL in the foregoingExamples. Titrations were carried out using NaOH as described above. Inaddition, data was adjusted by subtracting the contribution of residualacetate buffer based on the determination of acetate content by SE-HPLCas described in, for instance, Example 3. FIG. 11, Panel A shows thetitration results. FIG. 11, Panel B shows the dependence of buffercapacity on the concentration of Ab-hB7R1 formulations before and aftersubtracting the contribution of residual acetate buffer. The resultsclearly show the self-buffering capacity of Ab-hB7RP1 in this pH range.At 60 mg/ml it provides approximately as much buffer capacity in this pHrange as 10 mM sodium acetate buffer.

Example 12 Ab-hB7RP1 Stability in Self-Buffering and ConventionallyBuffered Formulations at 4° C. and 29° C.

Ab-hB7RP1 was prepared as described in the foregoing Examples andformulated as described above, in self-buffering formulations and informulations using a conventional buffering agent, either acetate orglutamate. All formulations contained 60 mg/ml Ab-hB7RP1. The stabilityof the solution's pH and of the Ab-hB7RP1 in the solution was monitoredfor twenty-six weeks of storage at 4° C. or at 29° C. Stability wasmonitored by determining monomeric Ab-hB7RP1 in the formulations overthe time course of storage. The determination was made using SE-HPLC asdescribed above. The results are shown in FIG. 12. Panel A shows theresults for storage at 4° C. Panel B shows the results for storage at29° C. Ab-hB7RP1 was at least as stable in the self-buffered formulationat 4° C. as the conventionally buffered formulations. At 29° C. theself-buffered formulation was at least as stable as the conventionallybuffered formulations, and may have been slightly better from 10 weeksthrough the last time point.

Example 13 pH Stability of Self-Buffered Ab-hB7RP1 at 4° C. and 29° C.

Self-buffered Ab-hB7RP1 at 60 mg/ml was prepared as described in theforegoing Example. pH was monitored over the time course and at the sametemperatures as described therein. The results are shown in FIG. 13.

Example 14 Buffer Capacity of Ab-hCD22 Formulations—pH 4.0 to 6.0

Self-buffering formulations of Ab-hCD22 were prepared and titrated overthe range of pH 5.0 to 4.0 and the range of 5.0 to 6.0, as described forAb-hOPGL and Ab-hB7RP1 in the foregoing Examples. Buffer capacities werecalculated from the titration data, also as described above. Buffercapacity as a function of concentration is shown in FIG. 14 fur both pHranges. Panel A shows the buffer capacity of the Ab-hCD22 formulationsover the range of pH 5.0 to 4.0. Buffer capacity is linearally dependenton concentration, and an approximately 21 mg/ml formulation of Ab-hCD22has a buffer capacity equal to that of 10 mM sodium acetate buffer pH5.0, measured in the same way. Panel B shows the buffer capacity as afunction of concentration over the pH range 5.0 to 6.0. In this range ofpH an approximately 30 mg/ml formulation of Ab-hCD22 has a buffercapacity equal to that of 10 mM sodium acetate buffer pH 5.0, measuredin the same way.

Example 15 Titrations and Buffer Capacities of Ab-hIL4R Formulations—pH5.0 to 4.0

Self-buffering formulations of Ab-hIL4R were prepared in concentrationsof 1, 10, 25, and 90 mg/ml, as described for Ab-hOPGL in the foregoingExamples. Titrations were carried out using HCl as described above. FIG.15, Panel A shows the titration results. FIG. 15, Panel B shows thedependence of buffer capacity on the concentration of Ab-hIL4R. Theresults clearly show the self-buffering capacity of Ab-hIL4R in this pHrange. At approximately 75 mg/ml it provides as much buffer capacity inthis pH range as 10 mM sodium acetate pH 5.0, measured in the same way.

Example 16 Titrations and Buffer Capacities of Ab-hIL4R Formulations—pH5.0 to 6.0

Self-buffering formulations of Ab-hIL4R were prepared in concentrationsof 1, 10, 25, and 90 mg/ml, as described for Ab-hOPGL in the foregoingExamples. Titrations were carried out using NaOH as described above.FIG. 16, Panel A shows the titration results. FIG. 16, Panel B shows thedependence of buffer capacity on the concentration of Ab-hIL4R in thispH range. The results, clearly show the self-buffering capacity ofAb-hIL4R in this pH range. At approximately 90 mg/ml it provides as muchbuffer capacity in this pH range as 10 mM sodium acetate pH 5.0,measured in the same way.

Example 17 Ab-hIL4R and pH Stability in Acetate and Self-BufferedAb-hIL4R Formulations at 37° C.

Self-buffered and acetate buffered formulations of Ab-hIL4R at pH 5.0and 70 mg/ml were prepared as described above. pH and Ab-hIL4R stabilitywere monitored in the formulations for 4 weeks at 37° C. Ab-hIL4Rstability was monitored by SE-HPLC as described above. The results areshown in FIG. 17. Panel A shows that Ab-hIL4R is at least as stable inthe self-buffered formulation as in the sodium acetate bufferformulation. Panel B shows that pH in the self-buffered formulation isas stable as in the sodium acetate buffer formulation.

What is claimed:
 1. A formulation comprising: water; and Tositumomab,wherein Tositumomab is at a concentration between 20 and 200 mg/ml.